Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)-Establishment of the First EV Biobank for Polytraumatized Patients.

In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing-thawing cycles and different storage conditions (RT, 4/-20/-80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing-thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.

MACROPHAGE SWITCHING: POLARIZATION AND MOBILIZATION AFTER TRAUMA.

ABSTRACT: Introduction: Trauma alters the immune response in numerous ways, affecting both the innate and adaptive responses. Macrophages play an important role in inflammation and wound healing following injury. We hypothesize that macrophages mobilize from the circulation to the site of injury and secondary sites after trauma, with a transition from proinflammatory (M1) shortly after trauma to anti-inflammatory (M2) at later time points. Methods: C57Bl6 mice (n = 6/group) underwent a polytrauma model using cardiac puncture/hemorrhage, pseudofemoral fracture, and liver crush injury. The animals were killed at several time points: uninjured, 24 h, and 7 days. Peripheral blood mononuclear cells, spleen, liver nonparenchymal cells, and lung were harvested, processed, and stained for flow cytometry. Macrophages were identified as CD68 + ; M1 macrophages were identified as iNOS + ; M2 macrophages as arginase 1 + . Results: We saw a slight presence of M1 macrophages at baseline in peripheral blood mononuclear cells (6.6%), with no significant change at 24 h and 7 days after polytrauma. In contrast, the spleen has a larger population of M1 macrophages at baseline (27.7%), with levels decreasing at 24 h and 7 days after trauma (20.6% and 12.6%, respectively). A similar trend is seen in the lung where at baseline 14.9% of CD68 + macrophages are M1, with subsequent continual decrease reaching 8.7% at 24 h and 4.4% at 7 days after polytrauma. M1 macrophages in the liver represent 14.3% of CD68 + population in the liver nonparenchymal cells at baseline. This percentage increases to 20.8% after trauma and decreases at 7 days after polytrauma (13.4%). There are few M2 macrophages in circulating peripheral blood mononuclear cells and in spleen at baseline and after trauma. The percentage of M2 macrophages in the lungs remains constant after trauma (7.2% at 24 h and 9.2% at 7 days). In contrast, a large proportion of M2 macrophages are seen in the liver at baseline (36.0%). This percentage trends upward and reaches 45.6% acutely after trauma and drops to 21.4% at 7 days. The phenotypic changes in macrophages seen in the lungs did not correlate with a functional change in the ability of the macrophages to perform oxidative burst, with an increase from 2.0% at baseline to 22.1% at 7 days after polytrauma ( P = 0.0258). Conclusion: Macrophage phenotypic changes after polytrauma are noted, especially with a decrease in the lung M1 phenotype and a short-term increase in the M2 phenotype in the liver. However, macrophage function as measured by oxidative burst increased over the time course of trauma, which may signify a change in subset polarization after injury not captured by the typical macrophage phenotypes.

Biological Connection of Psychological Stress and Polytrauma under Intensive Care: The Role of Oxytocin and Hydrogen Sulfide.

This paper explored the potential mediating role of hydrogen sulfide (H2S) and the oxytocin (OT) systems in hemorrhagic shock (HS) and/or traumatic brain injury (TBI). Morbidity and mortality after trauma mainly depend on the presence of HS and/or TBI. Rapid "repayment of the O2 debt" and prevention of brain tissue hypoxia are cornerstones of the management of both HS and TBI. Restoring tissue perfusion, however, generates an ischemia/reperfusion (I/R) injury due to the formation of reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, pre-existing-medical-conditions (PEMC's) can aggravate the occurrence and severity of complications after trauma. In addition to the "classic" chronic diseases (of cardiovascular or metabolic origin), there is growing awareness of psychological PEMC's, e.g., early life stress (ELS) increases the predisposition to develop post-traumatic-stress-disorder (PTSD) and trauma patients with TBI show a significantly higher incidence of PTSD than patients without TBI. In fact, ELS is known to contribute to the developmental origins of cardiovascular disease. The neurotransmitter H2S is not only essential for the neuroendocrine stress response, but is also a promising therapeutic target in the prevention of chronic diseases induced by ELS. The neuroendocrine hormone OT has fundamental importance for brain development and social behavior, and, thus, is implicated in resilience or vulnerability to traumatic events. OT and H2S have been shown to interact in physical and psychological trauma and could, thus, be therapeutic targets to mitigate the acute post-traumatic effects of chronic PEMC's. OT and H2S both share anti-inflammatory, anti-oxidant, and vasoactive properties; through the reperfusion injury salvage kinase (RISK) pathway, where their signaling mechanisms converge, they act via the regulation of nitric oxide (NO).

Role of the C5a-C5a receptor axis in the inflammatory responses of the lungs after experimental polytrauma and hemorrhagic shock.

Singular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

Early dynamic orchestration of immunologic mediators identifies multiply injured patients who are tolerant or sensitive to hemorrhage.

BACKGROUND: Multiply injured patients (MIPs) are at risk of complications including infections, and acute and prolonged organ dysfunction. The immunologic response to injury has been shown to affect outcomes. Recent advances in computational capabilities have shown that early dynamic coordination of the immunologic response is associated with improved outcomes after trauma. We hypothesized that patients who were sensitive or tolerant of hemorrhage would demonstrate differences in dynamic immunologic orchestration within hours of injury. METHODS: We identified two groups of MIPs who demonstrated distinct clinical tolerance to hemorrhage (n = 10) or distinct clinical sensitivity to hemorrhage (n = 9) from a consecutive cohort of 100 MIPs. Hemorrhage was quantified by integrating elevated shock index values for 24 hours after injury (shock volume). Clinical outcomes were quantified by average Marshall Organ Dysfunction Scores from days 2 to 5 after injury. Shock-sensitive patients had high cumulative organ dysfunction after lower magnitude hemorrhage. Shock-tolerant (ST) patients had low cumulative organ dysfunction after higher magnitude hemorrhage. Computational methods were used to analyze a panel of 20 immunologic mediators collected serially over the initial 72 hours after injury. RESULTS: Dynamic network analysis demonstrated the ST patients had increased orchestration of cytokines that are reparative and protective including interleukins 9, 17E/25, 21, 22, 23, and 33 during the initial 0- to 8-hour and 8- to 24-hour intervals after injury. Shock-sensitive patients had delayed immunologic orchestration of a network of largely proinflammatory and anti-inflammatory mediators. Elastic net linear regression demonstrated that a group of five mediators could discriminate between shock-sensitive and ST patients. CONCLUSIONS: Preliminary evidence from this study suggests that early immunologic orchestration discriminates between patients who are notably tolerant or sensitive to hemorrhage. Early orchestration of a group of reparative/protective mediators was amplified in shock-tolerant patients. LEVEL OF EVIDENCE: Prospective clinical outcomes study, level III.

Structural alterations and inflammation in the heart after multiple trauma followed by reamed versus non-reamed femoral nailing.

BACKGROUND: Approximately 30,000 patients with blunt cardiac trauma are recorded each year in the United States. Blunt cardiac injuries after trauma are associated with a longer hospital stay and a poor overall outcome. Organ damage after trauma is linked to increased systemic release of pro-inflammatory cytokines and damage-associated molecular patterns. However, the interplay between polytrauma and local cardiac injury is unclear. Additionally, the impact of surgical intervention on this process is currently unknown. This study aimed to determine local cardiac immunological and structural alterations after multiple trauma. Furthermore, the impact of the chosen fracture stabilization strategy (reamed versus non-reamed femoral nailing) on cardiac alterations was studied. EXPERIMENTAL APPROACH: 15 male pigs were either exposed to multiple trauma (blunt chest trauma, laparotomy, liver laceration, femur fracture and haemorrhagic shock) or sham conditions. Blood samples as well as cardiac tissue were analysed 4 h and 6 h after trauma. Additionally, murine HL-1 cells were exposed to a defined polytrauma-cocktail, mimicking the pro-inflammatory conditions after multiple trauma in vitro. RESULTS: After multiple trauma, cardiac structural changes were observed in the left ventricle. More specifically, alterations in the alpha-actinin and desmin protein expression were found. Cardiac structural alterations were accompanied by enhanced local nitrosative stress, increased local inflammation and elevated systemic levels of the high-mobility group box 1 protein. Furthermore, cardiac alterations were observed predominantly in pigs that were treated by non-reamed intramedullary reaming. The polytrauma-cocktail impaired the viability of HL-1 cells in vitro, which was accompanied by a release of troponin I and HFABP. DISCUSSION: Multiple trauma induced cardiac structural alterations in vivo, which might contribute to the development of early myocardial damage (EMD). This study also revealed that reamed femoral nailing (reamed) is associated with more prominent immunological cardiac alterations compared to nailing without reaming (non-reamed). This suggests that the choice of the initial fracture treatment strategy might be crucial for the overall outcome as well as for any post-traumatic cardiac consequences.

Expression of High Mobility Group Box 1 Protein in a Polytrauma Model During Ground Transport and Simulated High-Altitude Evacuation.

BACKGROUND: We investigated the expression of high mobility group box 1 (HMGB1) protein in a combat-relevant polytrauma/ acute respiratory distress syndrome (ARDS) model. We hypothesized that systemic HMGB1 expression is increased after injury and during aeromedical evacuation (AE) at altitude. METHODS: Female Yorkshire swine (n =15) were anesthetized and cannulated with a 23Fr dual-lumen catheter. Venovenous extracorporeal life support (VV ECLS) was initiated via the right jugular vein and carried out with animals uninjured on day 1 and injured by bilateral pulmonary contusion on day 2. On both days, animals underwent transport and simulated AE. Systemic HMGB1 expression was measured in plasma by ELISA. Plasma-free Hb (pfHb) was measured with the use of spectrophotometric methods. RESULTS: Plasma HMGB1 on day 1 was transiently higher at arrival to the AE chambers, increased significantly after injury, reaching highest values at 8,000 ft on day 2, after which levels decreased but remained elevated versus baseline at each time point. pfHb decreased on day 1 at 30,000 ft and significantly increased on day 2 at 8,000 ft and postflight. CONCLUSIONS: Systemic HMGB1 demonstrated sustained elevation after trauma and altitude transport and may provide a useful monitoring capability during en route care.

Cardiac Glucose and Fatty Acid Transport After Experimental Mono- and Polytrauma.

OBJECTIVE: The aim of this study was to define the influence of trauma on cardiac glucose and fatty acid transport. The effects were investigated in vivo in a porcine mono- and polytrauma model and in vitro in human cardiomyocytes, which were treated simultaneously with different inflammatory substances, mimicking posttraumatic inflammatory conditions. METHODS AND RESULTS: In the porcine fracture- and polytrauma model, blood glucose concentrations were measured by blood gas analysis during an observation period of 72 h. The expression of cardiac glucose and fatty acid transporters in the left ventricle was determined by RT-qPCR and immunofluorescence. Cardiac and hepatic glycogen storage was examined. Furthermore, human cardiomyocytes were exposed to a defined trauma-cocktail and the expression levels of glucose- and fatty acid transporters were determined. Early after polytrauma, hyperglycemia was observed. After 48 and 72 h, pigs with fracture- and polytrauma developed hypoglycemia. The propofol demand significantly increased posttrauma. The hepatic glycogen concentration was reduced 72 h after trauma. Cardiac glucose and fatty acid transporters changed in both trauma models in vivo as well as in vitro in human cardiomyocytes in presence of proinflammatory mediators. CONCLUSIONS: Monotrauma as well as polytrauma changed the cardiac energy transport by altering the expression of glucose and fatty acid transporters. In vitro data suggest that human cardiomyocytes shift to a state alike myocardial hibernation preferring glucose as primary energy source to maintain cardiac function.

Contributing factors in the development of acute lung injury in a murine double hit model.

OBJECTIVES: Blunt chest (thoracic) trauma (TxT) is known to contribute to the development of secondary pulmonary complications. Of these, acute lung injury (ALI) is common especially in multiply injured patients and might not only be due to the direct trauma itself, but seems to be caused by ongoing and multifactorial inflammatory changes. Nevertheless, the exact mechanisms and contributing factors of the development of ALI following blunt chest trauma are still elusive. METHODS: 60 CL57BL/6N mice sustained either blunt chest trauma combined with laparotomy without further interventions or a double hit (DH) including TxT and cecal ligation puncture (CLP) after 24 h to induce ALI. Animals were killed either 6 or 24 h after the second procedure. Pulmonary expression of inflammatory mediators cxcl1, cxcl5, IL-1ß and IL-6, neutrophil infiltration and lung tissue damage using the Lung Injury Score (LIS) were determined. RESULTS: Next to a moderate increase in other inflammatory mediators, a significant increase in CXCL1, neutrophil infiltration and lung injury was observed early after TxT, which returned to baseline levels after 24 h. DH induced significantly increased gene expression of cxcl1, cxcl5, IL-1ß and IL-6 after 6 h, which was followed by the postponed significant increase in the protein expression after 24 h compared to controls. Neutrophil infiltration was significantly enhanced 24 h after DH compared to all other groups, and exerted a slight decline after 24 h. LIS has shown a significant increase after both 6 and 24 h compared to both control groups as well the late TxT group. CONCLUSION: Early observed lung injury with moderate inflammatory changes after blunt chest trauma recovered quickly, and therefore, may be caused by mechanical lung injury. In contrast, lung injury in the ALI group did not undergo recovery and is closely associated with significant changes of inflammatory mediators. This model may be used for further examinations of contributing factors and therapeutic strategies to prevent ALI.

Acute Inflammation in Traumatic Brain Injury and Polytrauma Patients Using Network Analysis.

INTRODUCTION: Traumatic brain injury (TBI) is associated with secondary injury to the central nervous system (CNS) via inflammatory mechanisms. The combination of polytrauma and TBI further exacerbates the inflammatory response to injury; however, combined injury phenomena have not been thoroughly studied. In this study, we examined the inflammatory differences between patients with TBI versus patients with polytrauma, but no TBI (polytrauma). We hypothesize that patients with TBI have a heightened early inflammatory response compared with polytrauma. METHODS: We conducted a single-center retrospective study of a cohort of patients with polytrauma, who were enrolled in the PROPPR study. These patients had blood samples prospectively collected at eight time points in the first 3 days of admission. Using radiological data to determine TBI, our polytrauma cohort was dichotomized into TBI (n = 30) or polytrauma (n = 54). Inflammatory biomarkers were measured using ELISA. Data across time were compared for TBI versus polytrauma groups using Wilcoxon rank-sum test. Network analysis techniques were used to systematically characterize the inflammatory responses at admission. RESULTS: Patients with TBI (51.6%) had a higher 30-day mortality compared with polytrauma (16.9%) (P <0.001). Expression levels of IL6, IL8, and CCL2 were elevated from the 2-h through 24-h time points, becoming significant at the 6-h time point (IL6, IL8, and CCL2; P <0.05) (). CSF3 showed a similar pattern, but did not attain significance. TBI and polytrauma networks underwent diverging trends from admission to the 6-h time point. CONCLUSION: Patients with TBI demonstrated upregulations in proinflammatory cytokines IL6, IL8, and CCL2. Utilizing informatics methods, we were able to identify temporal differences in network trends, as well as uncharacterized cytokines and chemokines in TBI. These data suggest TBI induces a distinct inflammatory response and pathologically heightened inflammatory response in the presence of polytrauma and may propagate worsened patient outcomes including mortality.

Differential fracture response to traumatic brain injury suggests dominance of neuroinflammatory response in polytrauma.

Polytraumatic injuries, specifically long bone fracture and traumatic brain injury (TBI), frequently occur together. Clinical observation has long held that TBI can accelerate fracture healing, yet the complexity and heterogeneity of these injuries has produced conflicting data with limited information on underlying mechanisms. We developed a murine polytrauma model with TBI and fracture to evaluate healing in a controlled system. Fractures were created both contralateral and ipsilateral to the TBI to test whether differential responses of humoral and/or neuronal systems drove altered healing patterns. Our results show increased bone formation after TBI when injuries occur contralateral to each other, rather than ipsilateral, suggesting a role of the nervous system based on the crossed neuroanatomy of motor and sensory systems. Analysis of the humoral system shows that blood cell counts and inflammatory markers are differentially modulated by polytrauma. A data-driven multivariate analysis integrating all outcome measures showed a distinct pathological state of polytrauma and co-variations between fracture, TBI and systemic markers. Taken together, our results suggest that a contralateral bone fracture and TBI alter the local neuroinflammatory state to accelerate early fracture healing. We believe applying a similar data-driven approach to clinical polytrauma may help to better understand the complicated pathophysiological mechanisms of healing.

Systemic release of heat-shock protein 27 and 70 following severe trauma.

Trauma represents a major cause of morbidity and mortality worldwide. The endogenous inflammatory response to trauma remains not fully elucidated. Pro-inflammation in the early phase is followed by immunosuppression leading to infections, multi-organ failure and mortality. Heat-shock proteins (HSPs) act as intracellular chaperons but exert also extracellular functions. However, their role in acute trauma remains unknown. The aim of this study was to evaluate serum concentrations of HSP 27 and HSP 70 in severely injured patients. We included severely injured patients with an injury severity score of at least 16 and measured serum concentration of both markers at admission and on day two. We found significantly increased serum concentrations of both HSP 27 and HSP 70 in severely injured patients. Concomitant thoracic trauma lead to a further increase of both HSPs. Also, elevated concentrations of HSP 27 and HSP 70 were associated with poor outcome in these patients. Standard laboratory parameters did not correlate with neither HSP 27, nor with HSP 70. Our findings demonstrate involvement of systemic release of HSP 27 and HSP 70 after severe trauma and their potential as biomarker in polytraumatized patients.

[Prognostic value of the dynamic changes in extra vascular lung water index and angiopoietin-2 in severe multiple trauma patients with acute respiratory distress syndrome].

OBJECTIVE: To observe the dynamic changes in extra vascular lung water index (EVLWI) and angiopoietin-2 (Ang-2) in severe multiple trauma patients with acute respiratory distress syndrome (ARDS), analyze the risk factor for short-term mortality, and to evaluate their prognostic values for prognosis. METHODS: A total of 54 severe multiple trauma patients with ARDS admitted to emergency intensive care unit (ICU) of the Affiliated Hospital of Guizhou Medical University from June 2014 to December 2018 were enrolled. The acute physiology and chronic health evaluation II (APACHE II), injury severity score (ISS) and oxygenation index (PaO2/FiO2), EVLWI [pulse-induced contour cardiac output (PiCCO) monitor] and plasma Ang-2 level [enzyme-linked immunosorbent assay (ELISA)] at 0 (immediately), 24, 48 and 72 hours after ICU admission, and the differences in PaO2/FiO2, EVLWI and Ang-2 between 0 hour and 72 hours (ΔPaO2/FiO2, ΔEVLWI, ΔAng-2) were calculated. The 28-day survival of patients was recorded, and the patients were divided into survival group and non-survival group. The differences in above mentioned parameters between the two groups were compared. Multivariate Logistic regression was used to analyze the independent risk factors associated with the prognosis. Receiver operating characteristic (ROC) curve was drawn to evaluate the prognostic values of ΔEVLWI and ΔAng-2 on the prognosis, and the Kaplan-Meier survival curve was plotted. RESULTS: 115 patients were enrolled in the final analysis, 72 survived in 28 days, 43 died, and the mortality rate was 37.4%. The APACHE II and ISS scores of the non-survival group were significantly higher than those of the survival group [APACHE II score: 25.7±2.7 vs. 20.6±2.2, ISS score: 22.1±3.1 vs. 18.1±2.1, both P < 0.05]. EVLWI and Ang-2 showed a gradual downwards tendency with the prolongation of the length of ICU stay in the survival group, but no significant change was found in the non-survival group. Parallel contour test showed that both P < 0.05, indicating that the curves between the two groups had different tendencies and were not parallel. The levels of EVLWI, Ang-2 and PaO2/FiO2 showed no statistical differences from 0 hour to 24 hours between the two groups, but EVLWI and Ang-2 in the non-survival group were significantly higher than those in the survival group from 48 hours on [EVLWI (mL/kg): 15.5±4.2 vs. 10.8±3.2, Ang-2 (ng/L): 352.7±51.2 vs. 237.9±42.8, both P < 0.05], and PaO2/FiO2 was significantly decreased [mmHg (1 mmHg = 0.133 kPa): 126.1±43.7 vs. 211.2±33.8, P < 0.05]. The ΔEVLWI and ΔAng-2 in the non-survival group were significantly lower than those in the survival group [ΔEVLWI (mL/kg): -0.9±6.1 vs. 3.1±6.4, ΔAng-2 (ng/L): -45.3±32.1 vs. 79.8±58.2, both P < 0.05], but ΔPaO2/FiO2 showed no significant difference as compared with the survival group (mmHg: 23.2±24.2 vs. -22.1±22.8, P > 0.05). Multivariate Logistic regression analysis demonstrated that ΔEVLWI [odds ratio (OR) = 2.811, 95% confidence interval (95%CI) = 1.232-3.161, P = 0.001], ΔAng-2 (OR = 2.204, 95%CI = 1.012-3.179, P = 0.001) and APACHE II (OR = 1.206, 95%CI = 1.102-1.683, P = 0.002) were independent risk factors for 28-day mortality of severe multiple trauma patients with ARDS. ROC curve analysis showed that the area under ROC curve (AUC) of ΔEVLWI for predicting 28-day prognosis of severe multiple trauma patients with ARDS was 0.832, which was higher than ΔAng-2 (AUC = 0.790) and APACHE II (AUC = 0.735). When the cut-off value of ΔEVLWI was 2.3 mL/kg, the sensitivity was 79.1%, and the specificity was 81.9%. Kaplan-Meier survival curve showed that the patients with ΔEVLWI > 2.3 mL/kg had a significantly higher 28-day cumulative survival rate as compared with the patients with ΔEVLWI ≤ 2.3 mL/kg (log-rank test: χ2 = 23.385, P = 0.000). CONCLUSIONS: ΔEVLWI and ΔAng-2 can be used as independent risk factors for 28-day mortality of severe multiple trauma patients with ARDS, and the predictive value of ΔEVLWI was better than Ang-2 and APACHE II. Dynamic observation of EVLWI could improve the accuracy of death forecasting for severe multiple trauma patients with ARDS.

Histone Deacetylase Inhibitors: A Novel Strategy in Trauma and Sepsis.

Trauma remains a leading cause of morbidity and mortality among all age groups in the United States. Hemorrhagic shock and traumatic brain injury (TBI) are major causes of preventable death in trauma. Initial treatment involves fluid resuscitation to improve the intravascular volume. Although crystalloids may provide volume expansion, they do not have any pro-survival properties. Furthermore, aggressive fluid resuscitation can provoke a severe inflammatory response and worsen clinical outcomes. Due to logistical constraints, however, definitive resuscitation with blood products is often not feasible in the prehospital setting-highlighting the importance of adjunctive therapies. In recent years, histone deacetylase inhibitors (HDACis) have shown promise as pharmacologic agents for use in both trauma and sepsis. In this review, we discuss the role of histone deacetylases (HDACs) and pharmacologic agents that inhibit them (HDACis). We also highlight the therapeutic effects and mechanisms of action of HDACis in hemorrhagic shock, TBI, polytrauma, and sepsis. With further investigation and translation, HDACis have the potential to be a high-impact adjunctive therapy to traditional resuscitation.

A Rise in Neutrophil Cell Size Precedes Organ Dysfunction After Trauma.

INTRODUCTION: Organ dysfunction remains a major cause of morbidity after trauma. The development of organ dysfunction is determined by the inflammatory response, in which neutrophils are important effector cells. A femoral fracture particularly predisposes for the development of organ dysfunction. This study investigated the chronologic relation between neutrophil characteristics and organ dysfunction in trauma patients with a femoral fracture. METHODS: Patients with a femoral fracture presenting at the University Medical Center Utrecht between 2007 and 2013 were included. Data of neutrophil characteristics from standard hematological analyzers were recorded on a daily basis until the 28th day of hospital stay or until discharge. Generalized Estimating Equations were used to compare outcome groups. RESULTS: In total 157 patients were analyzed, of whom 81 had polytrauma and 76 monotrauma. Overall mortality within 90 days was 6.4% (n = 10). Eleven patients (7.0%) developed organ dysfunction. In patients who developed organ dysfunction a significant increase in neutrophil count (P = 0.024), a significant increase in neutrophil cell size (P = 0.026), a significant increase in neutrophil complexity (P < 0.004), and a significant decrease in neutrophil lobularity (P < 0.001) were seen after trauma. The rise in neutrophil cell size preceded the clinical manifestation of organ dysfunction in every patient. CONCLUSION: Patients who develop organ dysfunction postinjury show changes in neutrophil characteristics before organ dysfunction becomes clinically evident. These findings regarding post-traumatic organ dysfunction may contribute to the development of new prognostic tools for immune-mediated complications in trauma patients. LEVEL OF EVIDENCE: Level II, etiologic study.

Effect of Long-Term Polytrauma on Ventilator-Induced Diaphragmatic Dysfunction in a Piglet Model.

INTRODUCTION: Mechanical ventilation is known to activate oxidative stress and proteolytic pathways in the diaphragm. Trauma by inducing inflammation and activating proteolytic pathways may potentiate the effects of mechanical ventilation on the diaphragm. In a blunt chest trauma with concomitant injuries we tested the hypothesis that trauma via inflammation further activates the proteolytic pathways and worsens atrophy in the diaphragm. MATERIAL AND METHODS: Piglets were separated into two groups and underwent 72 h of mechanical ventilation. One group received a polytrauma (PT) by unilateral femur fracture, blunt chest trauma with lung contusion, laparotomy with standardized liver incision, and a predefined hemorrhagic shock. The second mechanically ventilated group (MV) did not receive any trauma. A non-ventilated group (Con) served as control.Diaphragmatic fiber dimensions, Western Blot analyses of proteolytic pathways, and lipid peroxidation and messenger ribonucleic acid (mRNA) levels of cytokines and nuclear factor kappa b subunit p65 were measured. RESULTS: Active Caspase-3 was significantly increased in MV (P = 0.019), and in PT (P = 0.02) compared with Con. Nuclear factor kappa b subunit p65, was upregulated in PT (P = 0.010) compared with Con. IL-6 mRNA increased significantly in PT compared with Con (P = 0.0024) but did not differ between Con and MV. CONCLUSION: Trauma and mechanical ventilation induced proteolysis and atrophy in the diaphragm, but only polytrauma induced an inflammatory response in the diaphragm. The additional traumatic inflammatory stimulus did not increase the levels of the prementioned variables. These data underline that inflammation is not a major contributor to ventilator-induced diaphragmatic dysfunction. TRIAL REGISTRY NUMBER: AZ 84-02.04.2014.A265 (Landesamt für Natur-, Umwelt- und Verbraucherschutz, LANUV NRW, Germany).

Assessment of Coagulation Homeostasis in Blunt, Penetrating, and Thermal Trauma: Guidance for a Multicenter Systems Biology Approach.

INTRODUCTION: Provisioning care for traumatically injured patients makes conducting research very proximal to injury difficult. These studies also inherently have regulatory barriers to overcome. Here we outline a protocol for acute-phase enrollment of traumatically injured patients into a prospective observational clinical trial with precise and comprehensive sample acquisition in support of a systems biology approach to a research study. METHODS: Experts in trauma, burn, blood coagulation, computational biology, and integrative systems biology developed a prospective study that would capture the natural history of coagulation pathology after traumatic injury. Blood was sampled at admission and serial time points throughout hospitalization. Concurrently, demographic and outcomes data were recorded and on-site point-of-care testing was implemented. Protocols were harmonized across sites and sampling protocols validated through demonstration of feasibility and sample quality assurance testing. A novel data integration platform was developed to store, visualize, and enable large-scale analysis of empirical and clinical data. Regulatory considerations were also addressed in protocol development. RESULTS: A comprehensive Manual of Operations (MOO) was developed and implemented at 3 clinical sites. After regulatory approval, the MOO was followed to collect 5,348 longitudinal samples from 1,547 patients. All samples were collected, processed, and stored per the MOO. Assay results and clinical data were entered into the novel data management platform for analyses. CONCLUSION: We used an iterative, interdisciplinary process to develop a systematic and robust protocol for comprehensive assessment of coagulation in traumatically injured patients. This MOO can be a template for future studies in the acute setting.

Remote Intestinal Injury Early After Experimental Polytrauma and Hemorrhagic Shock.

Dysfunction of the gut-blood barrier plays an important role in many diseases, such as inflammatory bowel disease, hemorrhagic shock (HS), or burn injury. However, little is known about gut barrier dysfunction after hemodynamically instable polytrauma (PT). Therefore, we aimed to evaluate the effects of PT and HS on remote intestinal damage and barrier dysfunction, especially regarding the role of zonula occludens protein 1 (ZO-1) as an important tight junction protein.Male C57BL/6 mice were subjected to either PT (thorax trauma, closed head injury, soft tissue injury, and distal femoral fracture), 60 min of pressure-controlled HS (30 ±â€Š5 mmHg), or PT+HS, or sham procedures.Animals of all trauma groups showed an increase in abdominal girth and dilation of the intestine during the experimental period, which was largest in the PT+HS group. Increased blood-tissue permeability to albumin (assessed by Evans blue dye) was found in the HS group. Experimental groups showed a slight increase in plasma concentration of intestinal fatty acid binding protein and some intestinal damage was histologically detectable. Of note, PT+HS animals revealed significantly reduced expression of ZO-1 in intestinal epithelial cells. In an in-vitro model, stimulation of human colon epithelial cells with peptidoglycan, but not with lipopolysaccharide, resulted in elevated secretion of pro-inflammatory cytokines, reflecting inflammatory activity of the intestinal epithelium.Taken together, PT and HS lead to increased permeability of the gut-blood barrier. Bacterial components may lead to production of inflammatory and chemotactic mediators by gut epithelial cells, underlining the role of the gut as an immunologically active organ.

Distinct Dynamics of Stem and Progenitor Cells in Blood of Polytraumatized Patients.

Endogenously mobilized stem and progenitor cells (SPCs) or exogenously provided SPCs are thought to be beneficial for trauma therapy. However, still little is known about the synchronized dynamics of the number of SPCs in blood after severe injury and parameters like cytokine profiles that correlate with these numbers. We determined the number of hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and mesenchymal stem/stromal cells in peripheral blood (PB) 0 to 3, 8, 24, 48, and 120 h after polytrauma in individual patients (injury severity score ≥ 21). We found that the number of blood SPCs follows on average a synchronous, inverse bell-shaped distribution, with an increase at 0 to 3 h, followed by a strong decrease, with a nadir in SPC numbers in blood at 24 or 48 h. The change in numbers of SPCs in PB between 48 h and 120 h revealed two distinct patterns: Pattern 1 is characterized by an increase in the number of SPCs to a level higher than normal, pattern 2 is characterized by an almost absent increase in the number of SPCs compared to the nadir. Changes in the concentrations of the cytokines CK, MDC, IL-8, G-CSF Gro-α, VEGF, and MCP-1 correlated with changes in the number of SPCs in PB or were closely associated with Pattern 1 or Pattern 2. Our data provide novel rationale for investigations on the role of stem cell mobilization in polytraumatized patients and its likely positive impact on trauma outcome.

Effect of ß-glucan on serum levels of IL-12, hs-CRP, and clinical outcomes in multiple-trauma patients: a prospective randomized study.

BACKGROUND: Trauma is associated with a profound immunological dysfunction. This predisposes patients to infections and adverse outcomes. ß-glucan has been implicated in the initiation of anti-microbial immune response. The present study aimed to evaluate the effects of an enteral diet containing ß-glucan on serum levels of IL-12 and highly-sensitive C-reactive protein (hs-CRP), occurrence of infection, and clinical outcomes in critically ill multiple-trauma patients. METHODS: Forty multiple trauma patients requiring enteral nutrition for at least 10 days were randomly assigned to the intervention group (n=20) or the placebo group (n=20). The intervention group received a high-protein enteral diet providing 3 g ß-glucan, and the control group received a similar diet, except for 3 g of maltodextrin as a placebo. Serum levels of IL-12 and hs-CRP were measured on days 0, 10, and 21. RESULTS: The ß-glucan group showed significantly higher serum levels of IL-12 on day 21 compared to the control group. Infection frequency and duration of mechanical ventilation were significantly lower in the ß-glucan group. A significant difference was found in the Sequential Organ Failure Assessment (SOFA) score in favor of the ß-glucan group. No difference was found in the serum levels of hs-CRP, length of ICU stay, occurrence of infection, and mortality rates between the two groups. CONCLUSION: ß-glucan may increase serum levels of IL-12, shorten the duration of mechanical ventilation, and reduce organ failure in critically ill multiple-trauma patients.