Metabolic Tissue Swelling and Local Microcirculation in Splanchnic Artery Occlusion Shock: Implications for Critical Illness.

Trauma is a leading cause of death in the United States. Advancements in shock resuscitation have been disappointing because the correct upstream mechanisms of injury are not being targeted. Recently, significant advancements have been shown using new cell-impermeant molecules that work by transferring metabolic water from swollen ischemic cells to the capillary, which restores tissue perfusion by microcirculatory decompression. The rapid normalization of oxygen transfer improves resuscitation outcomes. Since poor resuscitation and perfusion of trauma patients also causes critical illness and sepsis and can be mimicked by ischemia-reperfusion of splanchnic tissues, we hypothesized that inadequate oxygenation of the gut during trauma drives development of later shock and critical illness. We further hypothesized that this is caused by ischemia-induced water shifts causing compression no-reflow. To test this, the superior mesenteric artery of juvenile anesthetized swine was occluded for 30 minutes followed by 8 hours of reperfusion to induce mild splanchnic artery occlusion (SAO) shock. One group received the impermeant polyethylene glycol 20,000 Da (PEG-20k) that prevents metabolic cell swelling, and the other received a lactated Ringer's vehicle. Survival doubled in PEG-20k-treated swine along with improved macrohemodynamics and intestinal mucosal perfusion. Villus morphometry and plasma inflammatory cytokines normalized with impermeants. Plasma endotoxin rose over time after reperfusion, and impermeants abolished the rise. Inert osmotically active cell impermeants like PEG-20k improve intestinal reperfusion injury, SAO shock, and early signs of sepsis, which may be due to early restoration of mucosal perfusion and preservation of the septic barrier by reversal of ischemic compression no-reflow. SIGNIFICANCE STATEMENT: Significant advancements in treating shock and ischemia have been disappointing because the correct upstream causes have not been targeted. This study supports that poor tissue perfusion after intestinal ischemia from shock is caused by capillary compression no-reflow secondary to metabolic cell and tissue swelling since selectively targeting this issue with novel polyethylene glycol 20,000 Da-based cell-impermeant intravenous solutions reduces splanchnic artery occlusion shock, doubles survival time, restores tissue microperfusion, and preserves gut barrier function.

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.

Medication Reconciliation and Patient Safety in Trauma: Applicability of Existing Strategies.

The Joint Commission has established medication reconciliation as a National Patient Safety Goal, but it has not been studied much in trauma even though it is integral to safe patient care. This article reviews the existing medication reconciliation strategies and their applicability to the trauma setting. To perform medication reconciliation, hospitals use a variety of strategies including pharmacists or pharmacy technicians, electronic medical record tools, and patient-centered strategies. All of these strategies are limited in trauma. Subpopulations such as injured children, the elderly, and those with brain trauma are particularly challenging and are at risk for suboptimal care from inaccurate medication reconciliation. Further research is necessary to create a safe and efficient system for trauma patients.

Effects of Polyethylene Glycol-20k on Postresuscitation Myocardial and Cerebral Function in a Rat Model of Cardiopulmonary Resuscitation.

OBJECTIVES: Polyethylene glycol-20k is a hybrid cell impermeant that reduces ischemia injury and improves microcirculatory flow during and following low flow states through nonenergy-dependent water transfer in the microcirculation. We investigated the effects of polyethylene glycol-20k on postresuscitation microcirculation, myocardial and cerebral function, and duration of survival in a rat model of cardiopulmonary resuscitation. DESIGN: Ventricular fibrillation was induced in 20 male Sprague Dawley rats and untreated for 6 minutes. Animals were randomized into two groups (n = 10 for each group): polyethylene glycol-20k and control. Polyethylene glycol-20k (10% solution in saline, 10% estimated blood volume) and vehicle (saline) were administered at the beginning of cardiopulmonary resuscitation by continuous IV infusion. Resuscitation was attempted after 8 minutes of cardiopulmonary resuscitation. SETTING: University-Affiliated Research Laboratory. SUBJECTS: Sprague Dawley Rats. INTERVENTIONS: Polyethylene glycol-20k. MEASUREMENTS AND MAIN RESULTS: Buccal microcirculation was measured at baseline, 1, 3, and 6 hours after return of spontaneous circulation using a side-stream dark-field imaging device. Myocardial function was measured by echocardiography at baseline and every hour postresuscitation for 6 hours. The animals were then returned to their cage and observed for an additional 72 hours. Neurologic Deficit Scores were recorded at 24, 48, and 72 hours after resuscitation. Postresuscitation ejection fraction, cardiac output, and myocardial performance index were significantly improved in animals treated with polyethylene glycol-20k (p < 0.05). Perfused buccal vessel density and microcirculatory flow index values were significantly higher at all time points in the polyethylene glycol-20k group compared with the control group. Postresuscitation cerebral function and survival rate were also significantly improved in animals that received polyethylene glycol-20k. CONCLUSIONS: Administration of polyethylene glycol-20k following cardiopulmonary resuscitation improves postresuscitation myocardial and cerebral function, buccal microcirculation, and survival in a rat model of cardiopulmonary resuscitation.

Low-Volume Resuscitation for Hemorrhagic Shock: Understanding the Mechanism of PEG-20k.

Hemorrhagic shock leads to cell and tissue swelling and no reflow from compressed capillaries. Cell impermeants, including polyethylene glycol-20,000 (PEG-20k), reverse ischemia-induced cell swelling, extend low-volume resuscitation (LVR) time after shock, and increase tolerance to the low-volume state. The purpose of this study was to explore the mechanisms of action of PEG-20k containing LVR solutions. We hypothesized that PEG-20k acts as both an oncotic agent and an impermeant in the microcirculation, which moves water out of the space and into the capillaries to affect peripheral capillary filling and enhanced perfusion during the low-volume state. Rats were hemorrhaged until arterial lactate reached 9-10 mM/liter. Then, saline-based LVR solutions containing various impermeant materials were administered (10% blood volume). The LVR times for these solutions were determined by measuring the amount of time required for plasma lactate to climb back to 9 to 10 mM after LVR administration (low-volume tolerance). Capillary blood flow was measured by colored microspheres, and blood volume was measured by fluorescein isothiocyanate-labeled albumin dilution. Gluconate (impermeant), albumin (colloid), and PEG-20k (hybrid) increased LVR time over saline by 4-, 3-, and 8-fold, respectively. The combination of impermeant + albumin produced a biologic effect that was similar to PEG-20k alone. Capillary blood flow and plasma volume were decreased after shock with saline LVR but increased with PEG-20k, relative to saline. These data are consistent with the hypothesis that PEG-20k may act by establishing multiple osmotic gradients in the microcirculation to drive cell-to-capillary water transfer during hypovolemic shock.

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.

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.

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.

Ezrin functionality and hypothermic preservation injury in LLC-PK1 cells.

Renal epithelial cells from donor kidneys are susceptible to hypothermic preservation injury, which is attenuated when they over express the cytoskeletal linker protein ezrin. This study was designed to characterize the mechanisms of this protection. Renal epithelial cell lines were created from LLC-PK1 cells, which expressed mutant forms of ezrin with site directed alterations in membrane binding functionality. The study used cells expressing wild type ezrin, T567A, and T567D ezrin point mutants. The A and D mutants have constitutively inactive and active membrane binding conformations, respectively. Cells were cold stored (4 °C) for 6-24 h and reperfused for 1h to simulate transplant preservation injury. Preservation injury was assessed by mitochondrial activity (WST-1) and LDH release. Cells expressing the active ezrin mutant (T567D) showed significantly less preservation injury compared to wild type or the inactive mutant (T567A), while ezrin-specific siRNA knockdown and the inactive mutant potentiated preservation injury. Ezrin was extracted and identified from purified mitochondria. Furthermore, isolated mitochondria specifically bound anti-ezrin antibodies, which were reversed with the addition of exogenous recombinant ezrin. Recombinant wild type ezrin significantly reduced the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium, suggesting ezrin may modify mitochondrial function. In conclusion, the cytoskeletal linker protein ezrin plays a significant role in hypothermic preservation injury in renal epithelia. The mechanisms appear dependent on the molecule's open configuration (traditional linker functionality) and possibly a novel mitochondrial specific role, which may include modulation of mPTP function or calcium sensitivity.

Effects of polyethylene glycol-20k IV solution on donor management in a canine model of donor brain death.

BACKGROUND: Traditionally, vasopressors and crystalloids have been used to stabilize brain dead donors; however, the use of crystalloid is fraught with complications. This study aimed to investigate the effectiveness of a newly developed impermeant solution, polyethylene glycol-20k IV solution (PEG-20k) for resuscitation and support of brain dead organ donors. METHODS: Brain death was induced in adult beagle dogs and a set volume of PEG-20k or crystalloid solution was given thereafter. The animals were then resuscitated over 16 h with vasopressors and crystalloid as necessary to maintain mean arterial pressure of 80-100 mmHg. The kidneys were procured and cold-stored for 24 h, after which they were analyzed using the isolated perfused kidney model. RESULTS: The study group required significantly less crystalloid volume and vasopressors while having less urine output and requiring less potassium supplementation than the control group. Though the two groups' mean arterial pressure and lactate levels were comparable, the study group's kidneys showed less preservation injury after short-term reperfusion indexed by decreased lactate dehydrogenase release and higher creatinine clearance than the control group. CONCLUSIONS: The use of polyethylene glycol-20k IV solution for resuscitating brain dead donors decreases cell swelling and improves intravascular volume, thereby improving end organ oxygen delivery before procurement and so preventing ischemia-reperfusion injury after transplantation.

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.

Cholangiocyte Epithelial to Mesenchymal Transition (EMT) is a potential molecular mechanism driving ischemic cholangiopathy in liver transplantation.

Donation after circulatory death (DCD) has expanded the donor pool for liver transplantation. However, ischemic cholangiopathy (IC) after DCD liver transplantation causes inferior outcomes. The molecular mechanisms of IC are currently unknown but may depend on ischemia-induced genetic reprograming of the biliary epithelium to mesenchymal-like cells. The main objective of this study was to determine if cholangiocytes undergo epithelial to mesenchymal transition (EMT) after exposure to DCD conditions and if this causally contributes to the phenotype of IC. Human cholangiocyte cultures were exposed to periods of warm and cold ischemia to mimic DCD liver donation. EMT was tested by assays of cell migration, cell morphology, and differential gene expression. Transplantation of syngeneic rat livers recovered under DCD conditions were evaluated for EMT changes by immunohistochemistry. Human cholangiocytes exposed to DCD conditions displayed migratory behavior and gene expression patterns consistent with EMT. E-cadherin and CK-7 expressions fell while N-cadherin, vimentin, TGFß, and SNAIL rose, starting 24 hours and peaking 1-3 weeks after exposure. Cholangiocyte morphology changed from cuboidal (epithelial) before to spindle shaped (mesenchymal) a week after ischemia. These changes were blocked by pretreating cells with the Transforming Growth Factor beta (TGFß) receptor antagonist Galunisertib (1 µM). Finally, rats with liver isografts cold stored for 20 hours in UW solution and exposed to warm ischemia (30 minutes) at recovery had elevated plasma bilirubin 1 week after transplantation and the liver tissue showed immunohistochemical evidence of early cholangiocyte EMT. Our findings show EMT occurs after exposure of human cholangiocytes to DCD conditions, which may be initiated by upstream signaling from autocrine derived TGFß to cause mesenchymal specific morphological and migratory changes.

Combined Therapy With Polyethylene Glycol-20k and MCC950 Preserves Post-Resuscitated Myocardial Function in a Rat Model of Cardiac Arrest and Cardiopulmonary Resuscitation.

Background To investigate the therapeutic potential of combined therapy with polyethylene glycol-20k (PEG-20k) and MCC950 on post-resuscitation myocardial function in a rat model of cardiac arrest. Methods and Results Thirty rats were randomized into 5 groups: Sham, Control, PEG-20k, MCC950, PEG-20k+ MCC950. Except for sham, animals were subjected to 6 minutes of ventricular fibrillation followed by 8 minutes cardiopulmonary resuscitation. Two milliliters PEG-20k was administered by intravenous injection coincident with the start of cardiopulmonary resuscitation; MCC950 (10 mg/kg), a highly selective NLRP3 inflammasome inhibitor, was delivered immediately after restoration of spontaneous circulation. Myocardial function, sublingual microcirculation, mitochondrial function, plasma cardiac troponin I, and interleukin-1ß, expression of proteins in SIRT1 (sirtuin 1)/PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and NLRP3 (the NOD-like receptor family protein 3) inflammasome pathways were evaluated. Following cardiopulmonary resuscitation, myocardial function was compromised with a significantly decreased cardiac output, ejection fraction, and increased myocardial performance index, cardiac troponin I. Sublingual microcirculation was disturbed with impaired perfused vessel density and microvascular flow index. Cardiac arrest reduced mitochondrial routine respiration, Complex I-linked respiration, respiratory control rates and oxidative phosphorylation coupling efficiency. PEG-20k or MCC950 alone restored mitochondrial respiratory function, restituted sublingual microcirculation, and preserved myocardial function, whereas a combination of PEG-20k and MCC950 further improved these aspects. PEG-20k restored the expression of SIRT1 and PGC-1α, and blunted activation of NLRP3 inflammasomes. MCC950 suppressed expression of cleaved-caspase-1/pro-caspase-1, ASC (apoptosis-associated speck-like protein), GSDMD [gasdermin d], and interleukin-1ß. Conclusions Combined therapy with PEG-20k and MCC950 is superior to either therapy alone for preserving post-resuscitated myocardial function, restituting sublingual microcirculation at restoration of spontaneous circulation at 6 hours. The responsible mechanisms involve upregulated expression of SIRT1/PGC1-α in tandem with inhibition of NLRP3 inflammasomes.

Polyethylene glycol-20k reduces post-resuscitation cerebral dysfunction in a rat model of cardiac arrest and resuscitation: A potential mechanism.

Out-of-hospital cardiac arrest (CA) is a leading cause of death in the United States. Severe post-resuscitation cerebral dysfunction is a primary cause of poor outcome. Therefore, we investigate the effects of polyethylene glycol-20k (PEG-20k), a cell impermeant, on post-resuscitation cerebral function. Thirty-two male Sprague-Dawley rats were randomized into four groups: 1) Control; 2) PEG-20k; 3) Sham control; 4) Sham with PEG-20k. To investigate blood brain barrier (BBB) permeability, ten additional rats were randomized into two groups: 1) CPR+Evans Blue (EB); 2) Sham+EB. Ventricular fibrillation was induced and untreated for 8 min, followed by 8 min of CPR, and resuscitation was attempted by defibrillation. Cerebral microcirculation was visualized at baseline, 2, 4 and 6 h after return of spontaneous circulation (ROSC). Brain edema was assessed by comparing wet-to-dry weight ratios after 6 h. S-100ß, NSE and EB concentrations were analyzed to determine BBB permeability damage. For results, Post-resuscitation cerebral microcirculation was impaired compared to baseline and sham control (p < 0.05). However, dysfunction was reduced in animals treated with PEG-20k compared to control (p < 0.05). Post-resuscitation cerebral edema as measured by wet-to-dry weight ratio was lower in PEG-20k compared to control (3.23 ±â€¯0.5 vs. 3.36 ±â€¯0.4, p < 0.05). CA and CPR increased BBB permeability and damaged neuronal cell with associated elevation of S-100ß sand NSE serum levels. PEG-20k administered during CPR improved cerebral microcirculation and reducing brain edema and injury.

High-resolution respirometry for evaluation of mitochondrial function on brain and heart homogenates in a rat model of cardiac arrest and cardiopulmonary resuscitation.

The physiology and physiopathology process of mitochondrial function following cardiac arrest remains poorly understood. We aimed to assess mitochondrial respiratory function on the heart and brain homogenates from cardiac arrest rats. The expression level of SIRT1/PGC-1α pathway was measured by immunoblotting. 30 rats were assigned to the CA group and the sham group. Rats of CA were subjected to 6 min of untreated ventricular fibrillation (VF) followed by 8 min of cardiopulmonary resuscitation (CPR). Mitochondrial respiratory function was compromised following CA and I/R injury, as indicated by CIL (451.46 ± 71.48 vs. 909.91 ± 5.51 pmol/min*mg for the heart and 464.14 ± 8.22 vs. 570.53 ± 56.33 pmol/min*mg for the brain), CI (564.04 ± 64.34 vs. 2729.52 ± 347.39 pmol/min*mg for the heart and 726.07 ± 85.78 vs. 1762.82 ± 262.04 pmol/min*mg for the brain), RCR (1.88 ± 0.46 vs. 3.57 ± 0.38 for the heart and 2.05 ± 0.19 vs. 3.49 ± 0.19, for the brain) and OXPHOS coupling efficiency (0.45 ± 0.11 vs. 0.72 ± 0.03 for the heart and 0.52 ± 0.05 vs. 0.71 ± 0.01 for the brain). However, routine respiration was lower in the heart and comparable in the brain after CA. CIV did not change in the heart but was enhanced in the brain. Furthermore, both SIRT1 and PGC-1α were downregulated concurrently in the heart and brain. The mitochondrial respiratory function was compromised following CA and I/R injury, and the major affected respiratory state is complex I-linked respiration. Furthermore, the heart and the brain respond differently to the global I/R injury after CA in mitochondrial respiratory function. Inhibition of the SIRT1/PGC-1α pathway may be a major contributor to the impaired mitochondrial respiratory function.

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.

Effects of Polyethylene Glycol-20k on Coronary Perfusion Pressure and Postresuscitation Myocardial and Cerebral Function in a Rat Model of Cardiac Arrest.

Background Epinephrine increases the rate of return of spontaneous circulation. However, it increases severity of postresuscitation myocardial and cerebral dysfunction and reduces duration of survival. We investigated the effects of aortic infused polyethylene glycol, 20 000 molecular weight (PEG-20k) during cardiopulmonary resuscitation on coronary perfusion pressure, postresuscitation myocardial and cerebral function, and duration of survival in a rat model of cardiac arrest. Methods and Results Twenty-four male rats were randomized into 4 groups: (1) PEG-20k, (2) epinephrine, (3) saline control-intravenous, and (4) saline control-intra-aortic. Cardiopulmonary resuscitation was initiated after 6 minutes of untreated ventricular fibrillation. In PEG-20k and Saline-A, either PEG-20k (10% weight/volume in 10% estimated blood volume infused over 3 minutes) or saline was administered intra-aortically after 4 minutes of precordial compression. In epinephrine and placebo groups, either epinephrine (20 µg/kg) or saline placebo was administered intravenously after 4 minutes of precordial compression. Resuscitation was attempted after 8 minutes of cardiopulmonary resuscitation. Sublingual microcirculation was measured at baseline and 1, 3, and 5 hours after return of spontaneous circulation. Myocardial function was measured at baseline and 2, 4, and 6 hours after return of spontaneous circulation. Neurologic deficit scores were recorded at 24, 48, and 72 hours after return of spontaneous circulation. Aortic infusion of PEG-20k increased coronary perfusion pressure to the same extent as epinephrine. Postresuscitation sublingual microcirculation, myocardial and cerebral function, and duration of survival were improved in PEG-20k (P<0.05) compared with epinephrine (P<0.05). Conclusions Aortic infusion of PEG-20k during cardiopulmonary resuscitation increases coronary perfusion pressure to the same extent as epinephrine, improves postresuscitation myocardial and cerebral function, and increases duration of survival in a rat model of cardiac arrest.