Expired But Not Yet Dead: Examining the Red Blood Cell Storage Lesion in Extended-Storage Whole Blood.

ABSTRACT: Whole blood is a powerful resuscitation strategy for trauma patients but has a shorter shelf life than other blood products. The red blood cell storage lesion in whole blood has not previously been investigated beyond the standard storage period. In the present study, we hypothesized that erythrocytes in stored whole blood exhibit similar aspects of the red blood cell storage lesion and that transfusion of extended storage whole blood would not result in a more severe inflammatory response after hemorrhage in a murine model. To test this hypothesis, we stored low-titer, O-positive, whole blood units, and packed red blood cells (pRBCs) for up to 42 days, then determined aspects of the red blood cell storage lesion. Compared with standard storage pRBCs, whole blood demonstrated decreased microvesicle and free hemoglobin at 21 days of storage and no differences in osmotic fragility. At 42 days of storage, rotational thromboelastometry demonstrated that clotting time was decreased, alpha angle was increased, and clot formation time and maximum clot firmness similar in whole blood as compared with pRBCs with the addition of fresh frozen plasma. In a murine model, extended storage whole blood demonstrated decreased microvesicle formation, phosphatidylserine, and cell-free hemoglobin. After hemorrhage and resuscitation, TNF-a, IL-6, and IL-10 were decreased in mice resuscitated with whole blood. Red blood cell survival was similar at 24 h after transfusion. Taken together, these data suggest that red blood cells within whole blood stored for an extended period of time demonstrate similar or reduced accumulation of the red blood cell storage lesion as compared with pRBCs. Further examination of extended-storage whole blood is warranted.

Direct Peritoneal Resuscitation Improves Survival in a Murine Model of Combined Hemorrhage and Burn Injury.

INTRODUCTION: Combined burn injury and hemorrhagic shock are a common cause of injury in wounded warfighters. Current protocols for resuscitation for isolated burn injury and isolated hemorrhagic shock are well defined, but the optimal strategy for combined injury is not fully established. Direct peritoneal resuscitation (DPR) has been shown to improve survival in rats after hemorrhagic shock, but its role in a combined burn/hemorrhage injury is unknown. We hypothesized that DPR would improve survival in mice subjected to combined burn injury and hemorrhage. MATERIALS AND METHODS: Male C57/BL6J mice aged 8 weeks were subjected to a 7-second 30% total body surface area scald in a 90°C water bath. Following the scald, mice received DPR with 1.5 mL normal saline or 1.5 mL peritoneal dialysis solution (Delflex). Control mice received no peritoneal solution. Mice underwent a controlled hemorrhage shock via femoral artery cannulation to a systolic blood pressure of 25 mm Hg for 30 minutes. Mice were then resuscitated to a target blood pressure with either lactated Ringer's (LR) or a 1:1 ratio of packed red blood cells (pRBCs) and fresh frozen plasma (FFP). Mice were observed for 24 hours following injury. RESULTS: Median survival time for mice with no DPR was 1.47 hours in combination with intravascular LR resuscitation and 2.08 hours with 1:1 pRBC:FFP. Median survival time significantly improved with the addition of intraperitoneal normal saline or Delflex. Mice that received DPR followed by 1:1 pRBC:FFP required less intravascular volume than mice that received DPR with LR, pRBC:FFP alone, and LR alone. Intraperitoneal Delflex was associated with higher levels of tumor necrosis factor alpha and macrophage inflammatory protein 1 alpha and lower levels of interleukin 10 and intestinal fatty acid binding protein. Intraperitoneal normal saline resulted in less lung injury 1 hour postresuscitation, but increased to similar severity of Delflex at 4 hours. CONCLUSIONS: After a combined burn injury and hemorrhage, DPR leads to increased survival in mice. Survival was similar with the use of normal saline or Delflex. DPR with normal saline reduced the inflammatory response seen with Delflex and delayed the progression of acute lung injury. DPR may be a valuable strategy in the treatment of patients with combined burn injury and hemorrhage.

Save it-don't waste it! Maximizing utilization of erythrocytes from previously stored whole blood.

BACKGROUND: Recent military and civilian experience suggests that fresh whole blood may be the preferred for treatment of hemorrhagic shock, but its use is limited by its 21-day shelf life. The red blood cell storage lesion and coagulation status of packed red blood cells (pRBCs) salvaged from expired whole blood are unknown. We hypothesized that pRBCs can be salvaged from previously stored whole blood. METHODS: Cold stored, low-titer, O-positive, nonleukoreduced, whole blood units were obtained at 21 days of storage. Erythrocytes were separated by centrifugation, resuspended in AS-3, and stored for 21 additional days as salvaged pRBCs. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared with pRBCs prepared at the time of donation and stored in AS-3 for 42 days (standard pRBCs). In additional experiments, murine pRBCs were prepared from expired whole blood units and compared with those stored under standard conditions. Mice underwent hemorrhage and resuscitation with standard and salvaged pRBC units, and serum cytokines and free hemoglobin were determined. RESULTS: There were no significant differences in microvesicle formation or cell-free hemoglobin concentration between salvaged and standard pRBCs. There was decreased Band-3 and increased phosphatidylserine in the salvaged units as well as greater osmotic fragility. Salvaged pRBCs maintained consistent clot firmness. After hemorrhage and resuscitation in a murine model, salvaged pRBCs did not demonstrate increased serum cytokine levels. CONCLUSION: Salvaged pRBCs from previously stored whole blood accumulate the red blood cell storage lesion in a similar fashion to standard pRBCs and maintain consistent coagulability when reconstituted with plasma. Salvaged pRBCs are not associated with an increased inflammatory response when used for resuscitation in a murine model. Salvaged pRBCs may be a viable product for utilization in the treatment of traumatic hemorrhagic shock.

Acid Sphingomyelinase Inhibition Mitigates Histopathological and Behavioral Changes in a Murine Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) can lead to the development of chronic traumatic encephalopathy as a result of neuronal phosphorylated tau (p-tau) protein aggregation and neuroinflammation. Acid sphingomyelinase (Asm) may also contribute to post-TBI neurodegenerative disorders. We hypothesized that Asm inhibition would ameliorate p-tau aggregation, neuroinflammation, and behavioral changes after TBI in a murine model. TBI was generated using a weight-drop method. Asm inhibition in wild-type mice was achieved with a single injection of amitriptyline 1 h after TBI. Genetic Asm ablation was achieved using Asm-deficient mice (Asm-/-). Thirty days after TBI, mice underwent behavioral testing with the forced swim test for symptoms of depression or were euthanized for neurohistological analysis. Neuroinflammation was quantified using the microglial markers, ionized calcium-binding adaptor molecule 1 and transmembrane protein 119. Compared to sham mice, TBI mice demonstrated increased hippocampal p-tau. Mice that received amitriptyline after TBI demonstrated decreased p-tau compared to mice that received a saline control. Further, post-TBI Asm-/- mice demonstrated lower levels of p-tau compared to wild-type mice. Though a decrease in neuroinflammation was observed at 1 month post-TBI, no change was demonstrated with mice treated with amitriptyline. Similarly, TBI mice were more likely to show depression compared to mice that received amitriptyline after TBI. Utilizing a weight-drop method to induce moderate TBI, we have shown that genetic deficiency or pharmacological inhibition of Asm prevented hippocampal p-tau aggregation 1 month after injury as well as decreased symptoms of depression. These findings highlight an opportunity to potentially reduce the long-term consequences of TBI.

IFNγ and TNFα mediate CCL22/MDC production in alveolar macrophages after hemorrhage and resuscitation.

Acute lung injury is a major complication of hemorrhagic shock and the required resuscitation with large volumes of crystalloid fluids and blood products. We previously identified a role of macrophage-derived chemokine (CCL22/MDC) pulmonary inflammation following hemorrhage and resuscitation. However, further details regarding the induction of CCL22/MDC and its precise role in pulmonary inflammation after trauma remain unknown. In the current study we used in vitro experiments with a murine alveolar macrophage cell line, as well as an in vivo mouse model of hemorrhage and resuscitation, to identify key regulators in CCL22/MDC production. We show that trauma induces expression of IFNγ, which leads to production of CCL22/MDC through a signaling mechanism involving p38 MAPK, NF-κB, JAK, and STAT-1. IFNγ also activates TNFα production by alveolar macrophages, potentiating CCL22/MDC production via an autocrine mechanism. Neutralization of IFNγ or TNFα with specific antibodies reduced histological signs of pulmonary injury after hemorrhage and reduced inflammatory cell infiltration into the lungs.

Oxygenation extremes after traumatic brain injury transiently affect coagulation.

INTRODUCTION: Trauma patients may become hypoxic or iatrogenically hyperoxic in the early post-injury period. While both extremes of oxygenation may be harmful following injury, the mechanism has yet to be elucidated. We hypothesized that hypoxia or hyperoxia would induce changes in coagulation, creating a secondary insult exacerbating the primary injury. MATERIALS AND METHODS: Mice underwent traumatic brain injury (TBI) or sham and were subsequently exposed to room air or brief hypoxia (15% FiO2) then sacrificed at intervals. Another cohort of uninjured mice underwent more prolonged hypoxia (10% FiO2) or hyperoxia (60% FiO2). Platelet function was evaluated by ADP- or ASPi-induced impedance aggregometry and viscoelastic coagulation parameters were assessed with rotational thromboelastometry. RESULTS: In uninjured mice, ADP-induced platelet aggregation was acutely increased after prolonged hypoxia, but this difference did not persist at 6 h. Hypoxic and hyperoxic TBI mice had increased ADP induced platelet aggregation at 1 h compared to the normoxia group. TBI mice demonstrated an early increased platelet aggregation after hypoxia or hyperoxia. Viscoelastic coagulation parameters were similar between hypoxic, hyperoxic and room air groups at 1 h and 6 h. However, ROTEM clotting time and clot formation time were prolonged and maximal clot firmness were lower in sham TBI/hypoxia mice at 24 h. CONCLUSION: Post-TBI hypoxia and hyperoxia resulted in transiently increased platelet aggregation in response to ADP, without lasting effecting on platelet function or coagulation. Hypoxia also induced delayed hypocoagulability after platelet aggregation normalized. Brief changes in oxygenation may temporally affect coagulation and platelet aggregation, which could contribute to physiologic secondary injury after trauma.

UCH-L1 is a Poor Serum Biomarker of Murine Traumatic Brain Injury After Polytrauma.

BACKGROUND: Several serum biomarkers have been studied to diagnose incidence and severity of traumatic brain injury (TBI), but a reliable biomarker in TBI has yet to be identified. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) has been proposed as a biomarker in clinical and preclinical studies, largely in the setting of isolated TBI or concussion. The aim of this study was to evaluate the performance of UCH-L1 as a serum biomarker in the setting of polytrauma and TBI. METHODS: Multiple variations of murine TBI and polytrauma models were used to evaluate serum biomarkers. The different models included TBI with and without hemorrhagic shock and resuscitation, isolated extremity vascular ligation, extremity ischemia/reperfusion, and blunt tail injury. Blood was drawn at intervals after injury, and serum levels of neuron-specific enolase, UCH-L1, creatine kinase, and syndecan-1 were evaluated by enzyme-linked immunosorbent assay. RESULTS: UCH-L1 levels were not significantly different between TBI, tail injury, and sham TBI. By contrast, neuron-specific enolase levels were increased in TBI mice compared with tail injury and sham TBI mice. UCH-L1 levels increased regardless of TBI status at 30 min and 4 h after hemorrhagic shock and resuscitation. In mice that underwent femoral artery cannulation followed by hemorrhagic shock/resuscitation, UCH-L1 levels were significantly elevated compared with shock sham mice at 4 h (3158 ± 2168 pg/mL, 4 h shock versus 0 ± 0 pg/mL, 4 h shock sham; P < 0.01) and at 24 h (3253 ± 2954 pg/mL, 24 h shock versus 324 ± 482 pg/mL, 24 h shock sham; P = 0.03). No differences were observed in UCH-L1 levels between the sham shock and the arterial ligation, vein ligation, or extremity ischemia/reperfusion groups at any time point. Similar to UCH-L1, creatine kinase was elevated only after shock compared with sham mice at 4, 24, and 72 h after injury. CONCLUSIONS: Our study demonstrates that UCH-L1 is not a specific marker for TBI but is elevated in models that induce central and peripheral nerve ischemia. Given the increase in UCH-L1 levels observed after hemorrhagic shock, we propose that UCH-L1 may be a useful adjunct in quantifying severity of shock or global ischemia rather than as a specific marker of TBI.

Effects of Early Altitude Exposure on the Open Abdomen After Laparotomy in Trauma.

INTRODUCTION: While damage control surgery and resuscitation techniques have revolutionized the care of injured service members who sustain severe traumatic hemorrhage, the physiologic and inflammatory consequences of hemostatic resuscitation and staged abdominal surgery in the face of early aeromedical evacuation (AE) have not been investigated. We hypothesized that post-injury AE with an open abdomen would have significant physiologic and inflammatory consequences compared to AE with a closed abdomen. MATERIALS AND METHODS: Evaluation of resuscitation and staged abdominal closure was performed using a murine model of hemorrhagic shock with laparotomy. Mice underwent controlled hemorrhage to a systolic blood pressure of 25 mmHg and received either no resuscitation, blood product resuscitation, or Hextend resuscitation to a systolic blood pressure of either 50 mmHg (partial resuscitation) or 80 mmHg (complete resuscitation). Laparotomies were either closed prior to AE (closed abdomens) or left open during AE (open abdomens) and subsequently closed. AE was simulated with a 1-hour exposure to a hypobaric hypoxic environment at 8,000 feet altitude. Mice were euthanized at 0, 4, or 24 hours following AE. Serum was collected and analyzed for physiologic variables and inflammatory cytokine levels. Samples of lung and small intestine were collected for tissue cytokine and myeloperoxidase analysis as indicators of intestinal inflammation. Survival curves were also performed. RESULTS: Unresuscitated mice sustained an 85% mortality rate from hemorrhage and laparotomy, limiting the assessment of the effect of simulated AE in these subgroups. Overall survival was similar among all resuscitated groups regardless of the presence of hypobaric hypoxia, type of resuscitation, or abdominal closure status. Simulated AE had no observed effects on acid/base imbalance or the inflammatory response as compared to ground level controls. All mice experienced both metabolic acidosis and an acute inflammatory response after hemorrhage and injury, represented by an initial increase in serum interleukin (IL)-6 levels. Furthermore, mice with open abdomens had an elevated inflammatory response with increased levels of serum IL-10, serum tumor necrosis factor alpha, intestinal IL-6, intestinal IL-10, and pulmonary myeloperoxidase. CONCLUSION: These results demonstrate the complex interaction of AE and temporary or definitive abdominal closure after post-injury laparotomy. Contrary to our hypothesis, we found that AE in those animals with open abdomens is relatively safe with no difference in mortality compared to those with closed abdomens. However, given the physiologic and inflammatory changes observed in animals with open abdomens, further evaluation is necessary prior to definitive recommendations regarding the safety or downstream effects of exposure to AE prior to definitive abdominal closure.

Microvesicles generated following traumatic brain injury induce platelet dysfunction via adenosine diphosphate receptor.

BACKGROUND: Traumatic brain injury (TBI) can result in an acute coagulopathy including platelet dysfunction that can contribute to ongoing intracranial hemorrhage. Previous studies have shown adenosine diphosphate (ADP)-induced platelet aggregation to be reduced after TBI. In addition, circulating microvesicles (MVs) are increased following TBI and have been shown to play a role in post-TBI coagulopathy and platelet function. We hypothesized that post-TBI MVs would affect platelet aggregation in a murine head injury model. METHODS: Moderate TBI was performed using a weight-drop method in male C57BL6 mice. Whole blood, plasma, MVs, and MV-poor plasma were isolated from blood collected 10 minutes following TBI and were mixed separately with whole blood from uninjured mice. Platelet aggregation was measured with Multiplate impedance platelet aggregometry in response to ADP. The ADP P2Y12 receptor inhibitor, R-138727, was incubated with plasma and MVs from TBI mice, and platelet inhibition was again measured. RESULTS: Whole blood taken from 10-minute post-TBI mice demonstrated diminished ADP-induced platelet aggregation compared with sham mice. When mixed with normal donor blood, post-TBI plasma and MVs induced diminished ADP-induced platelet aggregation compared with sham plasma and sham MVs. By contrast, the addition of post-TBI MV-poor plasma to normal blood did not change ADP-induced platelet aggregation. The observed dysfunction in post-TBI ADP platelet aggregation was prevented by the pretreatment of post-TBI plasma with R-138727. Treatment of post-TBI MVs with R-138727 resulted in similar findings of improved ADP-induced platelet aggregation compared with nontreated post-TBI MVs. CONCLUSION: Adenosine diphosphate-induced platelet aggregation is inhibited acutely following TBI in a murine model. This platelet inhibition is reproduced in normal blood by the introduction of post-TBI plasma and MVs. Furthermore, observed platelet dysfunction is prevented when post-TBI plasma and MVs are treated with an inhibitor of the P2Y12 ADP receptor. Clinically observed post-TBI platelet dysfunction may therefore be partially explained by the presence of the ADP P2Y12 receptor within post-TBI MVs. LEVEL OF EVIDENCE: Level III.

Variable saline resuscitation in a murine model of combined traumatic brain injury and haemorrhage.

BACKGROUND: Resuscitation strategies for combined traumatic brain injury (TBI) with haemorrhage in austere environments are not fully established. Our aim was to establish the effects of various saline concentrations in a murine model of combined TBI and haemorrhage, and identify an effective resuscitative strategy for the far-forward environment. METHODS: Male C57BL/6 mice underwent closed head injury and subjected to controlled haemorrhage to a systolic blood pressure of 25 mmHg via femoral artery cannulation for 60 min. Mice were resuscitated with a fixed volume bolus or variable volumes of fluid to achieve a systolic blood pressure goal of 80 mmHg with 0.9% saline, 3% saline, 0.1-mL bolus of 23.4% saline, or a 0.1-mL bolus of 23.4% saline followed by 0.9% saline (23.4+). RESULTS: 23.4% saline and 23.4+ resulted in higher mortality at 6 h compared to 0.9% saline. Use of 3% saline required less volume to achieve targeted resuscitation, did not affect survival, and did not exacerbate post-traumatic inflammation. While 23.4+ resuscitation utilized lower volume, it resulted in hypernatremia, azotemia, and elevated systemic pro-inflammatory cytokines. All groups except 3% saline demonstrated progression of neuron damage, with cerebral oedema highest with 0.9% saline. CONCLUSIONS: 3% saline demonstrated favourable balance of survival, blood pressure restoration, minimization of inflammation, and prevention of ongoing neurologic injury without contributing to significant physiologic derangements. 23.4% saline administration may not be appropriate in the setting of concomitant hypotension.

Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.

Platelet Function Changes in a Time-Dependent Manner Following Traumatic Brain Injury in a Murine Model.

Traumatic brain injury (TBI) results in systemic changes in coagulation and inflammation that contribute to post-traumatic morbidity and mortality. The potential interaction of platelets and pro-inflammatory cytokines in the modulation of coagulation, microthrombosis, and venous thromboembolic events after moderate TBI has not been determined. Using a murine model, we hypothesized that the degree of platelet-induced coagulation varies depending on the platelet aggregation agonist platelet-induced coagulation changes in a time-dependent manner following TBI, and changes in platelet-induced coagulation are mirrored by changes in the levels of circulating pro-inflammatory cytokines. An established weight-drop model was used to induce TBI in anesthetized mice. Blood samples were collected at intervals after injury for measurements of platelet count, serum fibrinogen, pro-inflammatory cytokines, and determination of soluble P-selectin levels. Thromboelastometry was used to evaluate changes in hemostasis. Platelet function was determined using whole blood impedance aggregometry. Ten minutes following TBI, adenosine diphosphate-induced platelet aggregation decreased as measured by platelet aggregometry. Despite no changes in platelet counts and serum fibrinogen, platelet aggregation, pro-inflammatory cytokines, and soluble P-selectin were increased at 6 h after TBI. Rotation thromboelastometry demonstrated increased maximal clot firmness at 6 h. Platelet function and coagulability returned to baseline levels 24 h following head injury. Our data demonstrate that after TBI, acute platelet dysfunction occurs followed by rebound platelet hyperaggregation. Alterations in post-TBI platelet aggregation are reflected in whole blood thromboelastometry and are temporally associated with the systemic pro-inflammatory response.

Impact of tranexamic acid on coagulation and inflammation in murine models of traumatic brain injury and hemorrhage.

BACKGROUND: Posttraumatic coagulopathy and inflammation can exacerbate secondary cerebral damage after traumatic brain injury (TBI). Tranexamic acid (TXA) has been shown clinically to reduce mortality in hemorrhaging and head-injured trauma patients and has the potential to mitigate secondary brain injury with its reported antifibrinolytic and antiinflammatory properties. We hypothesized that TXA would improve posttraumatic coagulation and inflammation in a murine model of TBI alone and in a combined injury model of TBI and hemorrhage (TBI/H). METHODS: An established murine weight drop model was used to induce a moderate TBI. Mice were administered intraperitoneal injections of 10 mg/kg TXA or equivalent volume of saline 10 min after injury. An additional group of mice was subjected to TBI followed by hemorrhagic shock using a pressure-controlled model. TBI/H mice were given intraperitoneal injections of TXA or saline during resuscitation. Blood was collected at intervals after injury to assess coagulation by rotational thromboelastometry (ROTEM) and inflammation by Multiplex cytokine analysis. Soluble P-selectin, a biomarker of platelet activation, and serum neuron-specific enolase, a biomarker of cerebral injury, were measured at intervals. Brain homogenates were analyzed for inflammatory changes by Multiplex enzyme-linked immunosorbent assay, and splenic tissue was collected for splenic cell population assessment by flow cytometry. RESULTS: There were no coagulation, serum or cerebral cytokine, P-selectin, or neuron-specific enolase differences between mice treated with TXA or saline after TBI. After the addition of hemorrhagic shock and resuscitation to TBI, TXA administration still did not affect coagulation parameters, systemic or cerebral inflammation, or platelet activation, as compared with saline alone. At 24 hours after TBI, mice given TXA demonstrated lower splenic total cell counts central memory CD8, effector CD8, B cell, and increased naive CD4 cell populations. By contrast, TXA did not affect splenic leukocyte populations after combined TBI/H. CONCLUSIONS: Despite clinical data suggesting a mortality benefit, TXA did not modulate coagulation, inflammation, or biomarker generation in either the TBI or TBI/H murine models. Administration of TXA after TBI altered splenic leukocyte populations, which may contribute to a change in posttraumatic immune status. Future studies should be done to investigate the role of TXA in the development of posttraumatic immunosuppression and risk of nosocomial infections.

Impact of Platelets and Platelet-Derived Microparticles on Hypercoagulability Following Burn Injury.

An acute burn induced coagulopathy develops after scald injury, which evolves into a subacute, hypercoagulable state. Microparticles, specifically platelet-derived MPs (PMPs), have been suggested as possible contributors. We first developed a model of burn-induced coagulopathy and then sought to investigate the role of platelets and PMPs in coagulation after burn. We hypothesized that changes in circulating platelet and PMP populations after injury would contribute to the post-burn, hypercoagulable state. A murine scald model with 28% TBSA full thickness burn injury was utilized and blood samples were collected at intervals after injury. Circulating MP populations, platelet counts, overall coagulation, and platelet function were determined. Burn injury led to hypercoagulability on post-burn day one (PBD1), which persisted 6 days after injury (PBD6). On PBD1, there was a significant decrease in platelet numbers and a decline in platelet contribution to clot formation with a concomitant increase in circulating procoagulant PMPs. On PBD6, there was a significant increase in platelet numbers and in platelet activation with no change in PMPs compared with sham. Further, on PBD1 decreased ADP-induced platelet activation was observed with a contrasting increase in ADP-induced platelet activation on PBD6. We therefore concluded that there was a temporal change in the mechanisms leading to a hypercoagulable state after scald injury, that PMPs are responsible for changes seen on PBD1, and finally that ADP-induced platelet activation was key to the augmented clotting mechanisms 6 days after burn.

Microparticles impact coagulation after traumatic brain injury.

BACKGROUND: The pathophysiology that drives the subacute hypercoagulable state commonly seen after traumatic brain injury (TBI) is not well understood. Alterations caused by TBI in platelet and microparticle (MP) numbers and function have been suggested as possible causes; however, the contributions of platelets and MPs are currently unknown. MATERIALS AND METHODS: A weight-drop technique of TBI using a murine model of moderate head injury was used. Blood was collected at intervals after injury. MP enumeration and characterization were performed using Nanoparticle Tracking Analysis, and platelet counts and coagulation parameters were determined using thromboelastometry. A MP procoagulant assay was used to compare activity between injured and sham mice. RESULTS: At 24 h after injury, there were no changes in circulating platelet numbers. However, there was a decrease in platelet contribution to clot formation. In contrast, there was a decline in circulating total MP numbers. When MPs from sham mice were added to the blood from head-injured animals, there was a normalization of platelet contribution to clot formation. Conversely, when MPs from TBI mice were added to sham blood, there was a significant decrease in platelet contribution to clot formation. Notably, there was an increase in MP procoagulant activity in head-injured mice. CONCLUSIONS: MPs generated after TBI likely contribute to altered coagulation after head injury and may play a key role in the development of a posttraumatic hypercoagulable state in TBI patients.

Macrophage-derived chemokine (CCL22) is a novel mediator of lung inflammation following hemorrhage and resuscitation.

Resuscitation of patients after hemorrhage often results in pulmonary inflammation and places them at risk for the development of acute respiratory distress syndrome. Our previous data indicate that macrophage-derived chemokine (MDC/CCL22) is elevated after resuscitation, but its direct role in this inflammatory response is unknown. Macrophage-derived chemokine signaling through the C-C chemokine receptor type 4 (CCR4) is implicated in other pulmonary proinflammatory conditions, leading us to hypothesize that MDC may also play a role in the pathogenesis of lung inflammation following hemorrhage and resuscitation. To test this, C57BL/6 mice underwent pressure-controlled hemorrhage followed by resuscitation with lactated Ringer's solution. Pulmonary inflammation and inflammatory cell recruitment were analyzed with histological staining, and serum- and tissue-level cytokines were measured by enzyme-linked immunosorbent assay. Pulmonary inflammation and cell recruitment following hemorrhage and resuscitation were associated with systemic MDC levels. Inhibition of MDC via injection of a specific neutralizing antibody prior to hemorrhage and resuscitation significantly reduced pulmonary levels of the chemotactic cytokines keratinocyte-derived chemokine and macrophage inflammatory proteins 2 and 1α, as well as inflammatory cell recruitment to the lungs. Intravenous administration of recombinant MDC prior to resuscitation augmented pulmonary inflammation and cell recruitment. Histological evaluation revealed the expression of CCR4 within the bronchial epithelium, and in vitro treatment of activated bronchial epithelial cells with MDC resulted in production and secretion of neutrophil chemokines. The present study identifies MDC as a novel mediator of lung inflammation after hemorrhage and resuscitation. Macrophage-derived chemokine neutralization may provide a therapeutic strategy to mitigate this inflammatory response.

Des-acyl-ghrelin (DAG) normalizes hyperlactacidemia and improves survival in a lethal rat model of burn trauma.

Critical illness, including burn injury, results in elevated plasma lactate levels. Dysregulation of PI3K/Akt signaling has been shown to play a predominant role in the inactivation of skeletal muscle PDC and, hence, in hyperlactacidemia in rat models of sepsis and endotoxemia. This observation, and our previous finding that DAG can reverse burn-induced skeletal muscle proteolysis through the activation of PI3K/Akt pathway, led us to hypothesize that DAG may also attenuate hyperlactacidemia in burn injury. Our investigations revealed that burn injury significantly elevated both skeletal muscle lactate production and plasma lactate levels. Moreover, this was accompanied in skeletal muscle by a 5-7 fold increase in mRNA expression of pyruvate dehydrogenase kinases (PDK) 2 and 4, and a ∼30% reduction in PDC activity. DAG treatment of burn rats completely normalized not only the mRNA expression of the PDKs and PDC activity, but also hyperlactacidemia within 24h of burn injury. DAG also normalized epinephrine-induced lactate production by isolated skeletal muscles from normal rats. Moreover, DAG also improved survival in a lethal rat model of burn trauma. These findings with DAG may have clinical implications because chances of survival for critically ill patients are greatly improved if plasma lactate levels are normalized within 24h of injury.

Phosphodiesterase (PDE) inhibitor torbafylline (HWA 448) attenuates burn-induced rat skeletal muscle proteolysis through the PDE4/cAMP/EPAC/PI3K/Akt pathway.

Treatment of rats after burn-injury with the cyclic AMP phosphodiesterase (PDE) inhibitor, torbafylline (also known as HWA 448) significantly reversed changes in rat skeletal muscle proteolysis, PDE4 activity, cAMP concentrations and mRNA expression of TNFα, IL-6, ubiquitin and E3 ligases. Torbafylline also attenuated muscle proteolysis during in vitro incubation, and this effect was blocked by the inhibitor Rp-cAMPS. Moreover, torbafylline significantly increased phospho-Akt levels, and normalized downregulated phospho-FOXO1 and phospho-4E-BP1 in muscle of burn rats. Similarly, torbafylline also normalized phosphorylation levels of Akt and its downstream elements in TNFα+IFNγ treated C2C12 myotubes. Torbafylline enhanced protein levels of exchange protein directly activated by cAMP (Epac) both in skeletal muscle of burn rats and in TNFα+IFNγ treated C2C12 myotubes. Pretreatment with a specific antagonist of PI3K or Epac significantly reversed the inhibitory effects of torbafylline on TNFα+IFNγ-induced MAFbx mRNA expression and protein breakdown in C2C12 myotubes. Torbafylline inhibits burn-induced muscle proteolysis by activating multiple pathways through PDE4/cAMP/Epac/PI3K/Akt.

Preinjury alcohol exposure attenuates the neuroinflammatory response to traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) initiates a neuroinflammatory response that increases the risk of TBI-related mortality. Acute alcohol intoxication at the time of TBI is associated with improved survival. Ethanol is recognized as a systemic immunomodulator that may also impart neuroprotection. The effects of alcohol on TBI-induced neuroinflammation, however, are unknown. We hypothesized that ethanol treatment prior to TBI may provide neuroprotection by diminishing the neuroinflammatory response to injury. MATERIALS AND METHODS: Mice underwent gavage with ethanol (EtOH) or water (H2O) prior to TBI. Animals were subjected to blunt TBI or sham injury (Sham). Posttraumatic rapid righting reflex (RRR) and apnea times were assessed. Cerebral and serum samples were analyzed by ELISA for inflammatory cytokine levels. Serum neuron-specific enolase (NSE), a biomarker of injury severity, was also measured. RESULTS: Neurologic recovery from TBI was more rapid in H2O-treated mice compared with EtOH-treated mice. However, EtOH/TBI mice had a 4-fold increase in RRR time compared with EtOH/Sham, whereas H2O/TBI mice had a 15-fold increase in RRR time compared with H2O/Sham. Ethanol intoxication at the time of TBI significantly increased posttraumatic apnea time. Preinjury EtOH treatment was associated with reduced levels of proinflammatory cytokines IL-6, KC, MCP-1, and MIP-1α post TBI. NSE was significantly increased post injury in the H2O/TBI group compared with H2O/Sham but was not significantly reduced by EtOH pretreatment. CONCLUSIONS: Alcohol treatment prior to TBI reduces the local neuroinflammatory response to injury. The decreased neurologic and inflammatory impact of TBI in acutely intoxicated patients may be responsible for improved clinical outcomes.

Simulated aeromedical evacuation does not affect systemic inflammation or organ injury in a murine model of hemorrhagic shock.

Hemorrhagic shock is a primary injury amongst combat casualties. Aeromedical evacuation (AE) of casualties exposes patients to a hypobaric, hypoxic environment. The effect of this environment on the host response to hemorrhagic shock is unknown. In the present study, we sought to determine the effect of simulated AE on systemic inflammation and organ injury using a murine model of hemorrhagic shock. Mice underwent femoral artery cannulation and were hemorrhaged for 60 minutes. Mice were then resuscitated with a 1:1 ratio of plasma:packed red blood cells. At 1 or 24 hours after resuscitation, mice were exposed to a 5-hour simulated AE or remained at ground level (control). Serum was analyzed for cytokine concentrations and organs were assessed for neutrophil accumulation and vascular permeability. Mice in the simulated AE groups demonstrated reduced arterial oxygen saturation compared to ground controls. Serum cytokine concentrations, neutrophil recruitment, and vascular permeability in the lung, ileum, and colon in the simulated AE groups were not different from the ground controls. Our results demonstrate that mice exposed to simulated AE following hemorrhagic shock do not exhibit worsened systemic inflammation or organ injury compared to controls. The data suggest that AE has no adverse effect on isolated hemorrhagic shock.