Benjamin Kautza

Changes in massive transfusion over time: an early shift in the right direction?

Background: Increasing evidence suggests that high fresh frozen plasma:packed red blood cell (FFP:PRBC) and platelet:PRBC (PLT:PRBC) transfusion ratios may prevent or reduce the morbidity associated with early coagulopathy which complicates massive transfusion (MT). We sought to characterize changes in resuscitation which have occurred over time in a cohort severely injured patients requiring MT. Methods: Data were obtained from a multicenter prospective cohort study evaluating outcomes in blunt injured adults with hemorrhagic shock. MT was defined as requiring ≥10 units PRBCs within 24 hours postinjury. Mean PRBC, FFP, and PLT requirements (per unit; 6 hours, 12 hours, and 24 hours) were determined over time (2004–2009). Sub-MT, those patients just below the threshold for MT, were defined as requiring ≥7 and <10 units PRBCs in the initial 24 hours. The percent of resuscitation given at 6 hours relative to 24 hours total (6 of 24%) was determined and compared across “early” (admission until December 2007) and “recent” (after December 2007) periods for each component. Results: Over the study time period (2004–2009) for the MT group (n = 526), initial base deficit and presenting international normalized ratio were unchanged, while Injury Severity Score was significantly higher. The percent of patients who required MT overall significantly decreased over time. No significant differences were found over time for six-hour, 12-hour, or 24-hour FFP:PRBC and PLT:PRBC transfusion ratios in MT patients. Sub-MT patients (n = 344) had significantly higher six-hour FFP:PRBC ratios and significantly higher six-hour,12-hour, and 24-hour PLT:PRBC ratios in the recent time period. The six h/24 h% total for FFP and PLT transfusion was significantly greater in the recent time period. (FFP: 54% vs.70%; p = 0.004 and PLT 46% vs. 61%; p = 0.048). Conclusion: In a severely injured cohort requiring MT, FFP:PRBC and PLT:PRBC ratios have not changed over time, whereas the rate of MT overall has significantly decreased. During the recent time period (after December 2007), significantly higher transfusion ratios and a greater percent of 6-hour/24-hour FFP and PLT were found in the sub-MT group, those patients just below the PRBC transfusion threshold definition of MT. These data suggest early, more aggressive attainment of high transfusions ratios may reduce the requirement for MT and may shift overall blood requirements below those which currently define MT. Further prospective evidence is required to verify these findings. Level of Evidence: II.

Adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation

BACKGROUND Mortality in sepsis is most often attributed to the development of multiple organ failure. In sepsis, inflammation-mediated endothelial activation, defined as a proinflammatory and procoagulant state of the endothelial cells, has been associated with severity of disease. Thus, the objective of this study was to test the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation limits inflammation and endothelium activation to protect against organ injury in sepsis. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is an adenosine monophosphate analog, has been used to upregulate activity of AMPK. Compound C is a cell-permeable pyrrazolopyrimidine compound that inhibits AMPK activity. METHODS Wild-type mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice were randomized to vehicle, AICAR, or compound C. Mouse kidney endothelial cells were used for in vitro experiments. Renal and liver function were determined by serum cystatin C, blood urea nitrogen (BUN), creatinine, and alanine aminotransferase. Serum cytokines were measured by enzyme-linked immunosorbent assay. Microvascular injury was determined using Evans blue dye and electron microscopy. Immunohistochemistry was used to measure protein levels of phospho-AMPK (p-AMPK), microtubule-associated protein 1A/1B-light chain 3 (LC3), and intracellular adhesion molecule. LC3 levels were used as a measure of autophagosome formation. RESULTS AICAR decreased liver and kidney injury induced by CLP and minimized cytokine elevation in vivo and in vitro. CLP increased renal and hepatic phosphorylation of AMPK and autophagic signaling as determined by LC3. Inhibition of AMPK with compound C prevented CLP-induced autophagy and exacerbated tissue injury. Additionally, CLP led to endothelial injury as determined by electron microscopy and Evans blue dye extravasation, and AICAR limited this injury. Furthermore, AICAR limited CLP and lipopolysaccharide (LPS)-induced upregulation of intracellular adhesion molecule in vivo and in vitro and decreased LPS-induced neutrophil adhesion in vitro. CONCLUSIONS In this model, activation of AMPK was protective, and AICAR minimized organ injury by decreasing inflammatory cytokines and endothelial activation. These data suggest that AMPK signaling influences sepsis or LPS-induced endothelial activation and organ injury.

Massive Transfusion: An Evidence-Based Review of Recent Developments

The design and implementation of massive transfusion protocols with ratio-based transfusion of blood and blood products are important and active areas of investigation. A significant yet controversial body of literature exists to support the use of hemostatic resuscitation in massive transfusion and new data to support the use of adjuncts, such as recombinant factor VIIa and tranexamic acid. We review the developments in massive transfusion research during the past 5 years, including protocol implementation, hemostatic resuscitation, the use of tranexamic acid, and goal-directed therapy for coagulopathy. Furthermore, we provide a level of evidence analysis of the data surrounding the use of component therapy and recombinant factor VIIa in massive transfusion, summary recommendations for the various agents of resuscitation, and new methods of goal-directed therapy.

Benzyl alcohol attenuates acetaminophen‐induced acute liver injury in a Toll‐like receptor‐4‐dependent pattern in mice

Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in industrialized countries. Understanding the mechanisms of APAP‐induced liver injury as well as other forms of sterile liver injury is critical to improve the care of patients. Recent studies demonstrate that danger signaling and inflammasome activation play a role in APAP‐induced injury. The aim of these investigations was to test the hypothesis that benzyl alcohol (BA) is a therapeutic agent that protects against APAP‐induced liver injury by modulation of danger signaling. APAP‐induced liver injury was dependent, in part, on Toll‐like receptor (TLR)9 and receptor for advanced glycation endproducts (RAGE) signaling. BA limited liver injury over a dose range of 135‐540 μg/g body weight or when delivered as a pre‐, concurrent, or post‐APAP therapeutic. Furthermore, BA abrogated APAP‐induced cytokines and chemokines as well as high‐mobility group box 1 release. Moreover, BA prevented APAP‐induced inflammasome signaling as determined by interleukin (IL)‐1β, IL‐18, and caspase‐1 cleavage in liver tissues. Interestingly, the protective effects of BA on limiting liver injury and inflammasome activation were dependent on TLR4 signaling, but not TLR2 or CD14. Cell‐type–specific knockouts of TLR4 were utilized to further determine the protective mechanisms of BA. These studies found that TLR4 expression specifically in myeloid cells (LyzCre‐tlr4−/−) were necessary for the protective effects of BA. Conclusion: BA protects against APAP‐induced acute liver injury and reduced inflammasome activation in a TLR4‐dependent manner. BA may prove to be a useful adjunct in the treatment of APAP and other forms of sterile liver injury. (Hepatology 2014;60:990–1002)

Carbon Monoxide Protects Against Hemorrhagic Shock and Resuscitation-Induced Microcirculatory Injury and Tissue Injury

ABSTRACT Traumatic injury is a significant cause of morbidity and mortality worldwide. Microcirculatory activation and injury from hemorrhage contribute to organ injury. Many adaptive responses occur within the microcirculatory beds to limit injury including upregulation of heme oxygenase (HO) enzymes, the rate-limiting enzymes in the breakdown of heme to carbon monoxide (CO), iron, and biliverdin. Here we tested the hypothesis that CO abrogates trauma-induced injury and inflammation protecting the microcirculatory beds. Methods: C57Bl/6 mice underwent sham operation or hemorrhagic shock to a mean arterial pressure of 25 mmHg for 120 minutes. Mice were resuscitated with lactated Ringer’s at 2× the volume of maximal shed blood. Mice were randomized to receive CO-releasing molecule or inactive CO-releasing molecule at resuscitation. A cohort of mice was pretreated with tin protoporphyrin-IX to inhibit endogenous CO generation by HOs. Primary mouse liver sinusoidal endothelial cells were cultured for in vitro experiments. Results: Carbon monoxide–releasing molecule protected against hemorrhagic shock/resuscitation organ injury and systemic inflammation and reduced hepatic sinusoidal endothelial injury. Inhibition of HO activity with tin protoporphyrin-IX exacerbated liver hepatic sinusoidal injury. Hemorrhagic shock/resuscitation in vivo or cytokine stimulation in vitro resulted in increased endothelial expression of adhesion molecules that was associated with decreased leukocyte adhesion in vivo and in vitro. Conclusions: Hemorrhagic shock/resuscitation is associated with endothelial injury. Heme oxygenase enzymes and CO are involved in part in diminishing this injury and may prove useful as a therapeutic adjunct that can be harnessed to protect against endothelial activation and damage.

Polymicrobial sepsis is associated with decreased hepatic oxidative phosphorylation and an altered metabolic profile

BACKGROUND Organ failure in sepsis accounts for significant mortality worldwide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus of the organ and organism. Adaptive responses in metabolism are critical to the recovery at the cellular level. The purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration and significant metabolic changes in the liver. METHODS C57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or an operation without CLP. Mice were euthanized from 0-24 h after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity were measured. Global biochemical profiles of 311 metabolites were performed at the 8-h time point (n = 8/group) and analyzed by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry platforms by Metabolon (Durham, North Carolina). The influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated. RESULTS CLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measured at 12 h. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every tricarboxylic acid cycle intermediate and derivative, suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids. CONCLUSIONS Sepsis results in a metabolic response and profile consistent with increased anaerobic respiration, which occurs prior to significant changes in hemodynamics. The metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.

“Management of blunt renal injury: what is new?”

The diagnosis, workup and management of blunt renal injury have evolved greatly over the past decades. Evaluation and management of blunt renal injury echoes the increasing success of nonoperative management in other blunt abdominal solid organ injury, such as liver and spleen. Decision-making difficulties still remain regarding the optimal imaging, grading and degree of interventional or operative exploration used. Increasingly, initial nonoperative management has gained acceptance and appears to be applicable even high-grade injuries. Emerging techniques in highly sensitive imaging as well as interventional angiography have allowed safe nonoperative management in the appropriate patient. This review will focus on the contemporary workup and management of blunt renal injury while focusing on some of the emerging literatures in regard to refined imaging and grading of injuries as well as techniques to increase the success of nonoperative management.

Inhaled Carbon Monoxide Protects against the Development of Shock and Mitochondrial Injury following Hemorrhage and Resuscitation

Aims Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock. The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation. Results Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P<0.01). Additionally, CO limited the development of shock as determined by arterial blood pH (7.25±0.06 vs. 7.05±0.05; P<0.05), lactate levels (7.2±5.1 vs 13.3±6.0; P<0.05), and base deficit (13±3.0 vs 24±3.1; P<0.05). A dose response of CO (25–500 ppm) demonstrated protection against HS/R lung and liver injury as determined by MPO activity and serum ALT, respectively. CO limited HS/R-induced increases in serum tumor necrosis factor-α and interleukin-6 levels as determined by ELISA (P<0.05 for doses of 100–500ppm). Furthermore, inhaled CO limited HS/R induced oxidative stress as determined by hepatic oxidized glutathione:reduced glutathione levels and lipid peroxidation. In porcine HS/R, CO did not influence hemodynamics. However, CO limited HS/R-induced skeletal muscle and platelet mitochondrial injury as determined by respiratory control ratio (muscle) and ATP-linked respiration and mitochondrial reserve capacity (platelets). Conclusion These preclinical studies suggest that inhaled CO can be a protective therapy in HS/R; however, further clinical studies are warranted.

Blue light reduces organ injury from ischemia and reperfusion

Significance It is well established that light regulates mammalian biology. And yet, we have been unable to define and thus harness the underlying mechanisms so as to apply them to alter the course of human disease. In this study we determine that the spectrum of light is a critical determinant of its effect on critical illness. We show that an acute and short (24 h) exposure to high-illuminance (1,400 lx) blue spectrum (peak 442 nm) light prior to ischemia/reperfusion (I/R) significantly attenuates the degree of organ injury. Our characterization of the biological mechanisms through which blue light beneficially alters the cellular response to I/R provides an opportunity to develop novel therapeutics for the prevention and treatment of many diseases. Evidence suggests that light and circadian rhythms profoundly influence the physiologic capacity with which an organism responds to stress. However, the ramifications of light spectrum on the course of critical illness remain to be determined. Here, we show that acute exposure to bright blue spectrum light reduces organ injury by comparison with bright red spectrum or ambient white fluorescent light in two murine models of sterile insult: warm liver ischemia/reperfusion (I/R) and unilateral renal I/R. Exposure to bright blue light before I/R reduced hepatocellular injury and necrosis and reduced acute kidney injury and necrosis. In both models, blue light reduced neutrophil influx, as evidenced by reduced myeloperoxidase (MPO) within each organ, and reduced the release of high-mobility group box 1 (HMGB1), a neutrophil chemotactant and key mediator in the pathogenesis of I/R injury. The protective mechanism appeared to involve an optic pathway and was mediated, in part, by a sympathetic (β3 adrenergic) pathway that functioned independent of significant alterations in melatonin or corticosterone concentrations to regulate neutrophil recruitment. These data suggest that modifying the spectrum of light may offer therapeutic utility in sterile forms of cellular injury.

Sirtuin 1 Agonist Minimizes Injury and Improves the Immune Response Following Traumatic Shock.

ABSTRACT Survival from traumatic injury requires a coordinated and controlled inflammatory and immune response. Mitochondrial and metabolic responses to stress have been shown to play a role in these inflammatory and immune responses. We hypothesized that increases in mitochondrial biogenesis via a sirtuin 1 agonist would decrease tissue injury and partially ameliorate the immunosuppression seen following trauma. C57Bl/6 mice were subjected to a multiple trauma model. Mice were pretreated with either 100 mg/kg per day of the sirtuin 1 agonist, Srt1720, via oral gavage for 2 days prior to trauma and extended until the day the animals were killed, or they were pretreated with peroxisome proliferator-activated receptor &ggr; coactivator 1&agr; (PGC1&agr;) siRNA via hydrodynamic tail vein injection 48 h prior to trauma. Markers for mitochondrial function and biogenesis were measured in addition to splenocyte proliferative capacity and bacterial clearance. Srt1720 was noted to improve mitochondrial biogenesis, mitochondrial function, and complex IV activity following traumatic injury (P < 0.05), whereas knockdown of PGC1&agr; resulted in exacerbation of mitochondrial dysfunction (P < 0.05). These changes in mitochondrial function were associated with altered severity of hepatic injury with significant reductions in serum alanine aminotransferase levels seen in mice treated with srt1720. Splenocyte proliferative capacity and intraperitoneal bacterial clearance were evaluated as markers for overall immune function following trauma-hemorrhage. Treatment with Srt1720 minimized the trauma-induced decreases in splenocyte proliferation (P < 0.05), whereas treatment with PGC1&agr; siRNA led to diminished bacterial clearance. The PGC1&agr; signaling pathway is an important regulator of mitochondrial function and biogenesis, which can potentially be harnessed to protect against hepatic injury and minimize the immunosuppression that is seen following trauma-hemorrhage.