REBOA for the Treatment of Blast Polytrauma: Zone 3 Provides Cerebral Perfusion, Attenuates Organ Dysfunction and Reperfusion Coagulopathy Compared to Zone 1 in a Swine Model.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a lifesaving therapy for hemorrhagic shock following pelvic/lower extremity injuries in military settings. However, Zone 1 aortic occlusion (AO; above the celiac artery), while providing brain/cardiac perfusion, may induce/worsen visceral ischemia and organ dysfunction. In contrast, AO Zone 3 (below the renal arteries) provides abdominal perfusion potentially minimizing ischemia/reperfusion injury. We hypothesized that, compared with AO Zone 1, AO Zone 3 provides neuro/cardioprotection while minimizing visceral ischemia and reperfusion coagulopathy after severe traumatic hemorrhage due to pelvic/lower extremity injuries. METHODS: Fifty-kilogram male Yorkshire swine underwent a blast polytrauma injury followed by a resuscitation protocol with randomization to no AO (No AO, n = 6) or AO with REBOA at Zone 1 (AO Zone 1; n = 6) or Zone 3 (AO Zone 3; n = 4). Vital signs and intracranial pressure (ICP) were monitored for 240 minutes. Citrate native and tissue plasminogen activator challenge thrombelastography, prothrombin time, creatinine, lipase, total bilirubin, troponin, and enzyme-linked immunosorbent assays protein levels were measured at set intervals. RESULTS: Both AO groups had significant increases in mean arterial pressure during aortic occlusion. All three groups had significant increases in ICP, but final ICP in the No AO group (26 ± 5.8 mm Hg) was significantly elevated compared with AO Zone 1 (17 ± 5.2 mm Hg) and AO Zone 3 (16 ± 4.2 mm Hg) ( p < 0.01). The final mean troponin in the No AO group (4.10 ± 5.67 ng/mL) was significantly higher than baseline (0.03 ± 0.02 ng/mL, p < 0.05), while the two AO groups had no significant changes ( p > 0.05). AO Zone 1 was the only group associated with hyperfibrinolysis ( p < 0.05) and significantly increased prothrombin time ( p < 0.05). Only AO Zone 1 group had significantly higher markers of organ damage. CONCLUSION: Compared with AO Zone 1, AO Zone 3 provided similar neuro/cardioprotection but with less organ dysfunction and coagulopathy. This study suggests that Zone 3 REBOA may be preferable over Zone 1 for treating military relevant blast polytrauma with minimal intra-abdominal and chest trauma, but further clinical investigation is warranted.

Hemorrhagic shock and tissue injury provoke distinct components of trauma-induced coagulopathy in a swine model.

INTRODUCTION: Tissue injury (TI) and hemorrhagic shock (HS) are the major contributors to trauma-induced coagulopathy (TIC). However, the individual contributions of these insults are difficult to discern clinically because they typically coexist. TI has been reported to release procoagulants, while HS has been associated with bleeding. We developed a large animal model to isolate TI and HS and characterize their individual mechanistic pathways. We hypothesized that while TI and HS are both drivers of TIC, they provoke different pathways; specifically, TI reduces time to clotting, whereas, HS decreases clot strength stimulates hyperfibrinolysis. METHODS: After induction of general anesthesia, 50 kg male, Yorkshire swine underwent isolated TI (bilateral muscle cutdown of quadriceps, bilateral femur fractures) or isolated HS (controlled bleeding to a base excess target of - 5 mmol/l) and observed for 240 min. Thrombelastography (TEG), calcium levels, thrombin activatable fibrinolysis inhibitor (TAFI), protein C, plasminogen activator inhibitor 1 (PAI-1), and plasminogen activator inhibitor 1/tissue-type plasminogen activator complex (PAI-1-tPA) were analyzed at pre-selected timepoints. Linear mixed models for repeated measures were used to compare results throughout the model. RESULTS: TI resulted in elevated histone release which peaked at 120 min (p = 0.02), and this was associated with reduced time to clot formation (R time) by 240 min (p = 0.006). HS decreased clot strength at time 30 min (p = 0.003), with a significant decline in calcium (p = 0.001). At study completion, HS animals had elevated PAI-1 (p = 0.01) and PAI-1-tPA (p = 0.04), showing a trend toward hyperfibrinolysis, while TI animals had suppressed fibrinolysis. Protein C, TAFI and skeletal myosin were not different among the groups. CONCLUSION: Isolated injury in animal models can help elucidate the mechanistic pathways leading to TIC. Our results suggest that isolated TI leads to early histone release and a hypercoagulable state, with suppressed fibrinolysis. In contrast, HS promotes poor clot strength and hyperfibrinolysis resulting in hypocoagulability.

Decrease in acute periprosthetic joint infections incidence with vancomycin-loaded calcium sulfate beads in patients with non-modifiable risk factors. A randomized clinical trial.

OBJECTIVES: The influence of local antibiotic therapy in orthopedic surgery remains unclear. In this trial, we evaluated the incidence of periprosthetic joint infections (PJI), after local or intravenous (IV) antibiotic prophylaxis. The aim of this intervention was to compare the PJI incidence in a population with non-modifiable risk factors after local prophylaxis with vancomycin-loaded calcium sulfate beads versus a control group. METHODS: A total of 83 subjects were evaluated, inclusion criteria included participants over 60 years of age, with at least one main risk factor for PJI who underwent total hip or knee joint replacement between June 2019 and May 2020. Cases were randomized, and the intervention group received local prophylactic antibiotic therapy with calcium sulfate beads impregnated with vancomycin; conventional IV prophylactic antibiotic therapy was administered for the control group. C-reactive protein (CRP) and erythrocyte sedimentation rate (ERS) serum biomarkers were analyzed on the day 5 and weeks 4, 8, and 12. When needed, the synovial fluid sample was obtained and cultured for the early acute PJI diagnosis. RESULTS: Acute PJI was found in 27 patients (67.5%) in the control group and 4 (9.3%) in the intervention group. The variable analysis identified that local prophylaxis with calcium sulfate beads reduces the incidence of acute knee or hip PJI in patients with non-modifiable risk factors compared to conventional prophylaxis (p < 0.0001) with a relative risk of 0.13 (CI:0.05-0.35). Length of hospital stay was also shorter in the intervention group at 4.6 days, compared to 15.25 days in the control group; p < 0.001. CONCLUSIONS: Local antibiotic prophylaxis in patients with non-modifiable risk factors undergoing hip or knee replacement reduces the incidence of acute PJI compared to IV antibiotics. CLINICAL TRIALS: NCT03976466 (clnicaltrials.gov) LEVEL OF EVIDENCE: II.

Zone 1 REBOA in a combat DCBI swine model does not worsen brain injury.

BACKGROUND: Zone 1 resuscitative endovascular balloon occlusion of the aorta has been recommended for refractory shock after a dismounted complex blast injury for the austere combat scenario. While resuscitative endovascular balloon occlusion of the aorta should enhance coronary perfusion, there is a potential risk of secondary brain injury due to loss of cerebral autoregulation. We developed a combat casualty relevant dismounted complex blast injury swine model to evaluate the effects of resuscitative endovascular balloon occlusion of the aorta zone I on intracranial pressure and cerebral edema. We hypothesized that zone 1 aortic occlusion with resuscitative endovascular balloon occlusion of the aorta would increase mean arterial pressure transmitted in excessive intracranial pressure, thereby worsening brain injury. METHODS: 50 kg male Yorkshire swine were subjected to a combination dismounted complex blast injury model consisting of blast traumatic brain injury (50 psi, ARA Mobile Shock Laboratory), tissue injury (bilateral femur fractures), and hemorrhagic shock (controlled bleeding to a base deficit goal of 10 mEq/L). During the shock phase, pigs were randomized to no aortic occlusion (n = 8) or to 30 minutes of zone 1 resuscitative endovascular balloon occlusion of the aorta (zone 1 aortic occlusion group, n = 6). After shock, pigs in both groups received a modified Tactical Combat Casualty Care-based resuscitation and were monitored for an additional 240 minutes until euthanasia/death for a total of 6 hours. Intracranial pressure was monitored throughout, and brains were harvested for water content. Linear mixed models for repeated measures were used to compare mean arterial pressure and intracranial pressure between zone 1 aortic occlusion and no aortic occlusion groups. RESULTS: After dismounted complex blast injury, the zone 1 group had a significantly higher mean arterial pressure during hemorrhagic shock compared to the control group (41.2 mm Hg vs 16.7 mm Hg, P = .002). During balloon occlusion, intracranial pressure was not significantly elevated in the zone 1 aortic occlusion group vs control, but intracranial pressure was significantly lower in the zone 1 group at the end of the observation period. In addition, the zone 1 aortic occlusion group did not have increased brain water content (zone 1 aortic occlusion: 3.95 ± 0.1g vs no aortic occlusion: 3.95 ± 0.3 g, P = .87). Troponin levels significantly increased in the no aortic occlusion group but did not in the zone 1 aortic occlusion group. CONCLUSION: Zone 1 aortic occlusion using resuscitative endovascular balloon occlusion of the aorta in a large animal dismounted complex blast injury model improved proximal mean arterial pressure while not significantly increasing intracranial pressure during balloon inflation. Observation up to 240 minutes postresuscitation did not show clinical signs of worsening brain injury or cardiac injury. These data suggest that in a dismounted complex blast injury swine model, resuscitative endovascular balloon occlusion of the aorta in zone 1 may provide neuro- and cardioprotection in the setting of blast traumatic brain injury. However, longer monitoring periods may be needed to confirm that the neuroprotection is lasting.

Association of acute and chronic workloads with injury risk in high-performance junior tennis players.

This study examined the association and predictive ability of several markers of internal workload on risk of injury in high-performance junior tennis players. Fifteen young, high-level tennis players (9 males, 6 females; age: 17.2 ± 1.1 years; height: 178.5 ± 8.7 cm; mass: 68.1 ± 4.8 kg) participated in this investigation. Data on injury epidemiology and internal workload during training were obtained for one competitive season. The session-rating of perceived exertion (s-RPE) was used to calculate internal workload markers in absolute (acute workload and chronic workload for 2-weeks, 3-weeks and 4-weeks) and relative terms (acute:chronic workload ratios [ACWR] for 2-weeks, 3-weeks and 4-weeks). Associations and diagnostic power for predicting tennis injuries were examined through generalised estimating equations and receiver operating characteristics analyses. During the season, a total of 40 injuries were recorded, corresponding to 3.5 injuries per 1000 h of tennis practice. The acute workload was highly associated with injury incidence (P=0.04), as injury risk increased by 1.62 times (95% CI: 1.01-2.62) for every increase of 1858.7 arbitrary units (AU) of the workload during the most recent training week. However, acute workload was a poor predictor of injury, and associations between injury and internal workload markers were weak (all P>0.05). These findings demonstrate an association between high values of acute workload and the risk of injury in high-level tennis players. However, a high acute workload is only one of the many factors associated with injury, and by itself, has low predictive ability for injury.

Examining the Effect of Hypertonic Saline Administered for Reduction of Intracranial Hypertension on Coagulation.

BACKGROUND: Hypertonic saline (23.4%, HTS) bolus administration is common practice for refractory intracranial hypertension, but its effects on coagulation are unknown. We hypothesize that 23.4% HTS in whole blood results in progressive impairment of coagulation in vitro and in vivo in a murine model of traumatic brain injury (TBI). STUDY DESIGN: For the in vitro study, whole blood was collected from 10 healthy volunteers, and citrated native thrombelastography was performed with normal saline (0.9%, NS) and 23.4% HTS in serial dilutions (2.5%, 5%, and 10%). For the in vivo experiment, we assessed the effects of 23.4% HTS bolus vs NS on serial thrombelastography and tail-bleeding times in a TBI murine model (n = 10 rats with TBI and 10 controls). RESULTS: For the in vitro work, clinically relevant concentrations of HTS (2.5% dilution) shortened time to clot formation and increased clot strength (maximum amplitude) compared with control and NS. With higher HTS dosing (5% and 10% blood dilution), there was progressive prolongation of time to clot formation, decreased angle, and decreased maximum amplitude. In the in vivo study, there was no significant difference in thrombelastography measurements or tail-bleeding times after bolus administration of 23.4% HTS compared with NS at 2.5% blood volume. CONCLUSIONS: At clinically relevant dilutions of HTS, there is a paradoxical shortening of time to clot formation and increase in clot strength in vitro and no significant effects in a murine TBI model. However, with excess dilution, caution should be exercised when using serial HTS boluses in TBI patients at risk for trauma-induced coagulopathy.

Selective organ ischaemia/reperfusion identifies liver as the key driver of the post-injury plasma metabolome derangements.

BACKGROUND: Understanding the molecular mechanisms in perturbation of the metabolome following ischaemia and reperfusion is critical in developing novel therapeutic strategies to prevent the sequelae of post-injury shock. While the metabolic substrates fueling these alterations have been defined, the relative contribution of specific organs to the systemic metabolic reprogramming secondary to ischaemic or haemorrhagic hypoxia remains unclear. MATERIALS AND METHODS: A porcine model of selected organ ischaemia was employed to investigate the relative contribution of liver, kidney, spleen and small bowel ischaemia/reperfusion to the plasma metabolic phenotype, as gleaned through ultra-high performance liquid chromatography-mass spectrometry-based metabolomics. RESULTS: Liver ischaemia/reperfusion promotes glycaemia, with increases in circulating carboxylic acid anions and purine oxidation metabolites, suggesting that this organ is the dominant contributor to the accumulation of these metabolites in response to ischaemic hypoxia. Succinate, in particular, accumulates selectively in response to the hepatic ischemia, with levels 6.5 times spleen, 8.2 times small bowel, and 6 times renal levels. Similar trends, but lower fold-change increase in comparison to baseline values, were observed upon ischaemia/reperfusion of kidney, spleen and small bowel. DISCUSSION: These observations suggest that the liver may play a critical role in mediating the accumulation of the same metabolites in response to haemorrhagic hypoxia, especially with respect to succinate, a metabolite that has been increasingly implicated in the coagulopathy and pro-inflammatory sequelae of ischaemic and haemorrhagic shock.

Trauma and hemorrhagic shock activate molecular association of 5-lipoxygenase and 5-lipoxygenase-Activating protein in lung tissue.

BACKGROUND: Post-traumatic lung injury following trauma and hemorrhagic shock (T/HS) is associated with significant morbidity. Leukotriene-induced inflammation has been implicated in the development of post-traumatic lung injury through a mechanism that is only partially understood. Postshock mesenteric lymph returning to the systemic circulation is rich in arachidonic acid, the substrate of 5-lipoxygenase (ALOX5). ALOX5 is the rate-limiting enzyme in leukotriene synthesis and, following T/HS, contributes to the development of lung dysfunction. ALOX5 colocalizes with its cofactor, 5-lipoxygenase-activating protein (ALOX5AP), which is thought to potentiate ALOX5 synthetic activity. We hypothesized that T/HS results in the molecular association and nuclear colocalization of ALOX5 and ALOX5AP, which ultimately increases leukotriene production and potentiates lung injury. MATERIALS AND METHODS: To examine these molecular interactions, a rat T/HS model was used. Post-T/HS tissue was evaluated for lung injury through both histologic analysis of lung sections and biochemical analysis of bronchoalveolar lavage fluid. Lung tissue was immunostained for ALOX5 and ALOX5AP with association and colocalization evaluated by fluorescence resonance energy transfer. In addition, rats undergoing T/HS were treated with MK-886, a known ALOX5AP inhibitor. RESULTS: ALOX5 levels increase and ALOX5/ALOX5AP association occurred after T/HS, as evidenced by increases in total tissue fluorescence and fluorescence resonance energy transfer signal intensity, respectively. These findings coincided with increased leukotriene production and with the histological changes characteristic of lung injury. ALOX5/ALOX5AP complex formation, leukotriene production, and lung injury were decreased after inhibition of ALOX5AP with MK-886. CONCLUSIONS: These results suggest that the association of ALOX5/ALOX5AP contributes to leukotriene-induced inflammation and predisposes the T/HS animal to lung injury.

All animals are equal but some animals are more equal than others: Plasma lactate and succinate in hemorrhagic shock-A comparison in rodents, swine, nonhuman primates, and injured patients.

BACKGROUND: Plasma levels of lactate and succinate are predictors of mortality in critically injured patients in military and civilian settings. In relative terms, these metabolic derangements have been recapitulated in rodent, swine, and nonhuman primate models of severe hemorrhage. However, no direct absolute quantitative comparison has been evaluated across these species. METHODS: Ultra-high pressure liquid chromatography-mass spectrometry with stable isotope standards was used to determine absolute concentrations of baseline and postshock levels of lactate and succinate in rats, pigs, macaques, and injured patients. RESULTS: Baseline levels of lactate and succinate were most comparable to humans in macaques, followed by pigs and rats. Baseline levels of lactate in pigs and baseline and postshock levels of lactate and succinate in rats were significantly higher than those measured in macaques and humans. Postshock levels of lactate and succinate in pigs and macaques, respectively, were directly comparable to measurements in critically injured patients. CONCLUSION: Acknowledging the caveats associated with the variable degrees of shock in the clinical cohort, our data indicate that larger mammals represent a better model than rodents when investigating metabolic derangements secondary to severe hemorrhage.

The Metabolopathy of Tissue Injury, Hemorrhagic Shock, and Resuscitation in a Rat Model.

INTRODUCTION: The metabolic consequences of trauma induce significant clinical pathology. In this study, we evaluate the independent, metabolic contributions of tissue injury (TI) and combined tissue injury and hemorrhagic shock (TI/HS) using mass spectrometry (MS) metabolomics in a controlled animal model of critical injury. METHODS: Sprague-Dawley rats (n = 14) underwent TI alone or TI/HS, followed by resuscitation with normal saline and shed blood. Plasma was collected (baseline, post-laparotomy, post-HS, post-resuscitation) for ultra-high pressure liquid chromatography MS-metabolomics. Repeated-measures ANOVA with Tukey multiple column comparison test compared the fold change of metabolite concentration among the animal groups at corresponding time points. RESULTS: Four hundred forty metabolites were identified. TI alone did not change the metabolite levels versus baseline. TI/HS induced changes in metabolites from glycolysis, the tricarboxylic acid cycle, the pentose phosphate, fatty acid and glutathione homeostasis pathways, sulfur metabolism, and urea cycle versus TI alone. Following resuscitation many metabolites normalized to TI alone levels, including lactate, most tri-carboxylic acid metabolites, most urea cycle metabolites, glutathione disulfide, and some metabolites from both the pentose phosphate pathway and sulfur metabolism. CONCLUSIONS: Significant changes occur immediately following TI/HS versus TI alone. These metabolic changes are not explained by dilution as a number of metabolites remained unchanged or even increased following resuscitation. The differential metabolic changes resulting from TI alone and TI/HS provide foundation for future investigations severe injury in humans, where TI and HS are often concurrent. This investigation provides a foundation to evaluate metabolic-related outcomes and design-targeted resuscitation strategies.

Thrombelastography indicates limitations of animal models of trauma-induced coagulopathy.

BACKGROUND: Thrombelastography (TEG) has been used to characterize the coagulation changes associated with injury and shock. Animal models developed to investigate trauma-induced coagulopathy (TIC) have failed to produce excessive bleeding. We hypothesize that a native TEG will demonstrate marked differences in humans compared with these experimental models, which explains the difficulties in reproducing a clinically relevant coagulopathy in animal models. METHODS: Whole blood was collected from 138 healthy human volunteers, 25 swine and 66 Sprague-Dawley rats before experimentation. Citrated native TEGs were conducted on each whole blood sample within 2 h of collection. The clot initiation (R-time, minutes), angle (degrees), maximum amplitude (MA; millimeter), and lysis 30 min after MA (LY30; percentage) were analyzed and contrasted between species with data represented as the median and 25th to 75th quartile range. Difference between species was conducted with a Kruskal-Wallis test with alpha adjusted with a Bonferroni correction for multiple comparisons (alpha = 0.016). RESULTS: Median R-time (clot initiation) was 14.65 min (IQR: 13.2-16.3 min) for humans, 5.7 min (4.9-8.8) for pigs, and 5.2 min (4.4-6) for rodents. Humans had longer R-times than both pigs (P < 0.0001) and rats (P < 0.0001); pigs were not different from rats (P = 0.4439). Angle (fibrin cross-linking) was 42.3° (interquartile range [IQR]: 37.5-50.2) for humans, 71.7° (64.3-75.6) for pigs, and 61.8° (56.8-66.7) for rats. Humans had reduced angle compared with both pigs (P < 0.0001) and rats (P < 0.0001); pigs were not different from rats (P = 0.6052). MA (clot strength) was 55.5 mm (IQR: 52.0-59.5) for humans, 72.5 mm (70.4-75.5) for pigs, and 66.5 mm (56.5-68.6) for rats. Humans had reduced MA compared with both pigs (P < 0.0001) and rats (P < 0.0001); pigs were not different from rats (P = 0.0161). LY30 (fibrinolysis) was 1.5% (IQR: 0.975-2.5) for humans, 3.3% (1.9-4.3) for pigs, and 0.5% (0.1-1.2) for rats. Humans had a lesser LY30 than pigs (P = 0.0062) and a greater LY30 than rats (P < 0.0001), and pigs had a greater LY30 than rats (P < 0.0001). CONCLUSIONS: Humans, swine, and rodents have distinctly different coagulation systems, when evaluated by citrated native TEG. Animals are hypercoagulable with rapid clotting times and clots strengths nearly 50% stronger than humans. These coagulation differences indicate the limitations of previous models of trauma-induced coagulopathy in producing coagulation abnormalities associated with increased bleeding. The inherent hypercoagulable baseline tendencies of these animals may result in subclinical biochemical changes that are not detected by conventional TEG and should be taken into consideration when extrapolated to clinical medicine.

Hemorrhagic shock and tissue injury drive distinct plasma metabolome derangements in swine.

BACKGROUND: Tissue injury and hemorrhagic shock induce significant systemic metabolic reprogramming in animal models and critically injured patients. Recent expansions of the classic concepts of metabolomic aberrations in tissue injury and hemorrhage opened the way for novel resuscitative interventions based on the observed abnormal metabolic demands. We hypothesize that metabolic demands and resulting metabolic signatures in pig plasma will vary in response to isolated or combined tissue injury and hemorrhagic shock. METHODS: A total of 20 pigs underwent either isolated tissue injury, hemorrhagic shock, or combined tissue injury and hemorrhagic shock referenced to a sham protocol (n = 5/group). Plasma samples were analyzed by UHPLC-MS. RESULTS: Hemorrhagic shock promoted a hypermetabolic state. Tissue injury alone dampened metabolic responses in comparison to sham and hemorrhagic shock, and attenuated the hypermetabolic state triggered by shock with respect to energy metabolism (glycolysis, glutaminolysis, and Krebs cycle). Tissue injury and hemorrhagic shock had a more pronounced effect on nitrogen metabolism (arginine, polyamines, and purine metabolism) than hemorrhagic shock alone. CONCLUSION: Isolated or combined tissue injury and hemorrhagic shock result in distinct plasma metabolic signatures. These findings indicate that optimized resuscitative interventions in critically ill patients are possible based on identifying the severity of tissue injury and hemorrhage.

Red blood cells in hemorrhagic shock: a critical role for glutaminolysis in fueling alanine transamination in rats.

Red blood cells (RBCs) are the most abundant host cell in the human body and play a critical role in oxygen transport and systemic metabolic homeostasis. Hypoxic metabolic reprogramming of RBCs in response to high-altitude hypoxia or anaerobic storage in the blood bank has been extensively described. However, little is known about the RBC metabolism following hemorrhagic shock (HS), the most common preventable cause of death in trauma, the global leading cause of total life-years lost. Metabolomics analyses were performed through ultra-high pressure liquid chromatography-mass spectrometry on RBCs from Sprague-Dawley rats undergoing HS (mean arterial pressure [MAP], <30 mm Hg) in comparison with sham rats (MAP, >80 mm Hg). Steady-state measurements were accompanied by metabolic flux analysis upon tracing of in vivo-injected 13C15N-glutamine or inhibition of glutaminolysis using the anticancer drug CB-839. RBC metabolic phenotypes recapitulated the systemic metabolic reprogramming observed in plasma from the same rodent model. Results indicate that shock RBCs rely on glutamine to fuel glutathione (GSH) synthesis and pyruvate transamination, whereas abrogation of glutaminolysis conferred early mortality and exacerbated lactic acidosis and systemic accumulation of succinate, a predictor of mortality in the military and civilian critically ill populations. Glutamine is here identified as an essential amine group donor in HS RBCs, plasma, liver, and lungs, providing additional rationale for the central role glutaminolysis plays in metabolic reprogramming and survival following severe hemorrhage.

Glutamine metabolism drives succinate accumulation in plasma and the lung during hemorrhagic shock.

BACKGROUND: Metabolomic investigations have consistently reported succinate accumulation in plasma after critical injury. Succinate receptors have been identified on numerous tissues, and succinate has been directly implicated in postischemic inflammation, organ dysfunction, platelet activation, and the generation of reactive oxygen species, which may potentiate morbidity and mortality risk to patients. Metabolic flux (heavy-isotope labeling) studies demonstrate that glycolysis is not the primary source of increased plasma succinate during protracted shock. Glutamine is an alternative parent substrate for ATP generation during anaerobic conditions, a biochemical mechanism that ultimately supports cellular survival but produces succinate as a catabolite. We hypothesize that succinate accumulation during hemorrhagic shock is driven by glutaminolysis. METHODS: Sprague-Dawley rats were subjected to hemorrhagic shock for 45 minutes (shock, n = 8) and compared with normotensive shams (sham, n = 8). At 15 minutes, animals received intravenous injection of C5-N2-glutamine solution (iLG). Blood, brain, heart, lung, and liver tissues were harvested at defined time points. Labeling distribution in samples was determined by ultrahigh-pressure liquid chromatography-mass spectrometry metabolomic analysis. Repeated-measures analysis of variance with Tukey comparison determined significance of relative fold change in metabolite level from baseline. RESULTS: Hemorrhagic shock instigated succinate accumulation in plasma and lungs tissues (8.5- vs. 1.1-fold increase plasma succinate level from baseline, shock vs. sham, p = 0.001; 3.2-fold higher succinate level in lung tissue, shock vs. sham, p = 0.006). Metabolomic analysis identified labeled glutamine and labeled succinate in plasma (p = 0.002) and lung tissue (p = 0.013), confirming glutamine as the parent substrate. Kinetic analyses in shams showed constant total levels of all metabolites without significant change due to iLG. CONCLUSION: Glutamine metabolism contributes to increased succinate concentration in plasma during hemorrhagic shock. The glutaminolytic pathway is implicated as a therapeutic target to prevent the contribution of succinate accumulation in plasma and the lung-to-postshock pathogenesis.

Plasma First Resuscitation Reduces Lactate Acidosis, Enhances Redox Homeostasis, Amino Acid and Purine Catabolism in a Rat Model of Profound Hemorrhagic Shock.

The use of aggressive crystalloid resuscitation to treat hypoxemia, hypovolemia, and nutrient deprivation promoted by massive blood loss may lead to the development of the blood vicious cycle of acidosis, hypothermia, and coagulopathy and, utterly, death. Metabolic acidosis is one of the many metabolic derangements triggered by severe trauma/hemorrhagic shock, also including enhanced proteolysis, lipid mobilization, as well as traumatic diabetes. Appreciation of the metabolic benefit of plasma first resuscitation is an important concept. Plasma resuscitation has been shown to correct hyperfibrinolysis secondary to severe hemorrhage better than normal saline. Here, we hypothesize that plasma first resuscitation corrects metabolic derangements promoted by severe hemorrhage better than resuscitation with normal saline. Ultra-high-performance liquid chromatography-mass spectrometry-based metabolomics analyses were performed to screen plasma metabolic profiles upon shock and resuscitation with either platelet-free plasma or normal saline in a rat model of severe hemorrhage. Of the 251 metabolites that were monitored, 101 were significantly different in plasma versus normal saline resuscitated rats. Plasma resuscitation corrected lactate acidosis by promoting glutamine/amino acid catabolism and purine salvage reactions. Plasma first resuscitation may benefit critically injured trauma patients by relieving the lactate burden and promoting other non-clinically measured metabolic changes. In the light of our results, we propose that plasma resuscitation may promote fueling of mitochondrial metabolism, through the enhancement of glutaminolysis/amino acid catabolism and purine salvage reactions. The treatment of trauma patients in hemorrhagic shock with plasma first resuscitation is likely not only to improve coagulation, but also to promote substrate-specific metabolic corrections.

Shock-induced systemic hyperfibrinolysis is attenuated by plasma-first resuscitation.

BACKGROUND: We developed a hemorrhagic shock animal model to replicate an urban prehospital setting where resuscitation fluids are limited to assess the effect of saline versus plasma in coagulopathic patients. An in vitro model of whole blood dilution with saline exacerbated tissue plasminogen activator (tPA)-mediated fibrinolysis, while plasma dilution did not change fibrinolysis. We hypothesize that shock-induced hyperfibrinolysis can be attenuated by resuscitation with plasma while exacerbated by saline. METHODS: Sprague-Dawley rats were hemorrhaged to a mean arterial pressure of 25 mm Hg and maintained in shock for 30 minutes. Animals were resuscitated with either normal saline (NS) or platelet-free plasma (PFP) with a 10% total blood volume bolus, followed by an additional 5 minutes of resuscitation with NS to increase blood pressure to a mean arterial pressure of 30 mm Hg. Animals were observed for 15 minutes for the assessment of hemodynamic response and survival. Blood samples were analyzed with thrombelastography paired with protein analysis. RESULTS: The median percentage of total blood volume shed per group were similar (NS, 52.5% vs. PFP, 55.7; p = 0.065). Survival was 50% in NS compared with 100% in PFP. The change in LY30 and tPA levels from baseline to shock was similar between groups (LY30 PFP, 10; interquartile range [IQR], 4.3-11.2; NS, 4.5; IQR, 4.1-14.2; p = 1.00; tPA PFP, 16.6 ng/mL; IQR, 13.7-27.8; NS, 22.4; IQR, 20.1-25.5; p = 0.240). After resuscitation, the median change in LY30 was greater in the NS group (13.5; IQR, 3.5-19.9) compared with PFP (-4.9%; IQR, -9.22 to 0.25 p = 0.004), but tPA levels did not significantly change (NS, 1.4; IQR, -6.2 to 7.1 vs. PFP, 1.7; IQR, -5.2 to 6.8; p = 0.699). CONCLUSION: Systemic hyperfibrinolysis is driven by hypoperfusion and associated with increased levels of tPA. Plasma is a superior resuscitation fluid to NS in a prehospital model of severe hemorrhagic shock as it attenuates hyperfibrinolysis and improves systemic perfusion.

Trauma/hemorrhagic shock instigates aberrant metabolic flux through glycolytic pathways, as revealed by preliminary (13)C-glucose labeling metabolomics.

BACKGROUND: Metabolic derangement is a key hallmark of major traumatic injury. The recent introduction of mass spectrometry-based metabolomics technologies in the field of trauma shed new light on metabolic aberrations in plasma that are triggered by trauma and hemorrhagic shock. Alteration in metabolites associated with catabolism, acidosis and hyperglycemia have been identified. However, the mechanisms underlying fluxes driving such metabolic adaptations remain elusive. METHODS: A bolus of U-(13)C-glucose was injected in Sprague-Dawley rats at different time points. Plasma extracts were analyzed via ultra-high performance liquid chromatography-mass spectrometry to detect quantitative fluctuations in metabolite levels as well as to trace the distribution of heavy labeled carbon isotopologues. RESULTS: Rats experiencing trauma did not show major plasma metabolic aberrations. However, trauma/hemorrhagic shock triggered severe metabolic derangement, resulting in increased glucose levels, lactate and carboxylic acid accumulation. Isotopologue distributions in late Krebs cycle metabolites (especially succinate) suggested a blockade at complex I and II of the electron transport chain, likely due to mitochondrial uncoupling. Urate increased after trauma and hemorrhage. Increased levels of unlabeled mannitol and citramalate, metabolites of potential bacterial origin, were also observed in trauma/hemorrhagic shock rats, but not trauma alone or controls. CONCLUSIONS: These preliminary results are consistent with observations we have recently obtained in humans, and expand upon our early results on rodent models of trauma and hemorrhagic shock by providing the kinetics of glucose fluxes after trauma and hemorrhage. Despite the preliminary nature of this study, owing to the limited number of biological replicates, results highlight a role for shock, rather than trauma alone, in eliciting systemic metabolic aberrations. This study provides the foundation for tracing experiments in rat models of trauma. The goal is to improve our understanding of substrate specific metabolic derangements in trauma/hemorrhagic shock, so as to design resuscitative strategies tailored toward metabolic alterations and the severity of trauma.

Fibrinolysis shutdown phenotype masks changes in rodent coagulation in tissue injury versus hemorrhagic shock.

INTRODUCTION: Systemic hyperfibrinolysis (accelerated clot degradation) and fibrinolysis shutdown (impaired clot degradation) are associated with increased mortality compared with physiologic fibrinolysis after trauma. Animal models have not reproduced these changes. We hypothesize rodents have a shutdown phenotype that require an exogenous profibrinolytic to differentiate mechanisms that promote or inhibit fibrinolysis. METHODS: Fibrinolysis resistance was assessed by thrombelastography (TEG) using exogenous tissue plasminogen activator (tPA) titrations in whole blood. There were 3 experimental groups: (1) tissue injury (laparotomy/bowel crush), (2) shock (hemorrhage to mean arterial pressure of 20 mmHg), and (3) control (arterial cannulation and tracheostomy). Baseline and 30-minute postintervention blood samples were collected, and assayed with TEG challenged with taurocholic acid (TUCA). RESULTS: Rats were resistant to exogenous tPA; the percent clot remaining 30 minutes after maximum amplitude (CL30) at 150 ng/mL (P = .511) and 300 ng/mL (P = .931) was similar to baseline, whereas 600 ng/mL (P = .046) provoked fibrinolysis. Using the TUCA challenge, the percent change in CL30 from baseline was increased in tissue injury compared with control (P = .048.), whereas CL30 decreased in shock versus control (P = .048). tPA increased in the shock group compared with tissue injury (P = .009) and control (P = .012). CONCLUSION: Rats have an innate fibrinolysis shutdown phenotype. The TEG TUCA challenge is capable of differentiating changes in clot stability with rats undergoing different procedures. Tissue injury inhibits fibrinolysis, whereas shock promotes tPA-mediated fibrinolysis.

Early hemorrhage triggers metabolic responses that build up during prolonged shock.

Metabolic staging after trauma/hemorrhagic shock is a key driver of acidosis and directly relates to hypothermia and coagulopathy. Metabolic responses to trauma/hemorrhagic shock have been assayed through classic biochemical approaches or NMR, thereby lacking a comprehensive overview of the dynamic metabolic changes occurring after shock. Sprague-Dawley rats underwent progressive hemorrhage and shock. Baseline and postshock blood was collected, and late hyperfibrinolysis was assessed (LY30 >3%) in all of the tested rats. Extreme and intermediate time points were collected to assay the dynamic changes of the plasma metabolome via ultra-high performance liquid chromatography-mass spectrometry. Sham controls were used to determine whether metabolic changes could be primarily attributable to anesthesia and supine positioning. Early hemorrhage-triggered metabolic changes that built up progressively and became significant during sustained hemorrhagic shock. Metabolic phenotypes either resulted in immediate hypercatabolism, or late hypercatabolism, preceded by metabolic deregulation during early hemorrhage in a subset of rats. Hemorrhagic shock consistently promoted hyperglycemia, glycolysis, Krebs cycle, fatty acid, amino acid, and nitrogen metabolism (urate and polyamines), and impaired redox homeostasis. Early dynamic changes of the plasma metabolome are triggered by hemorrhage in rats. Future studies will determine whether metabolic subphenotypes observed in rats might be consistently observed in humans and pave the way for tailored resuscitative strategies.

Mesenteric lymph diversion abrogates 5-lipoxygenase activation in the kidney following trauma and hemorrhagic shock.

BACKGROUND: Early acute kidney injury (AKI) following trauma is associated with multiorgan failure and mortality. Leukotrienes have been implicated both in AKI and in acute lung injury. Activated 5-lipoxygenase (5-LO) colocalizes with 5-LO-activating protein (FLAP) in the first step of leukotriene production following trauma and hemorrhagic shock (T/HS). Diversion of postshock mesenteric lymph, which is rich in the 5-LO substrate of arachidonate, attenuates lung injury and decreases 5-LO/FLAP associations in the lung after T/HS. We hypothesized that mesenteric lymph diversion (MLD) will also attenuate postshock 5-LO-mediated AKI. METHODS: Rats underwent T/HS (laparotomy, hemorrhagic shock to a mean arterial pressure of 30 mm Hg for 45 minutes, and resuscitation), and MLD was accomplished via cannulation of the mesenteric duct. Extent of kidney injury was determined via histology score and verified by urinary neutrophil gelatinase-associated lipocalin assay. Kidney sections were immunostained for 5-LO and FLAP, and colocalization was determined by fluorescence resonance energy transfer signal intensity. The end leukotriene products of 5-LO were determined in urine. RESULTS: AKI was evident in the T/HS group by derangement in kidney tubule architecture and confirmed by neutrophil gelatinase-associated lipocalin assay, whereas MLD during T/HS preserved renal tubule morphology at a sham level. MLD during T/HS decreased the associations between 5-LO and FLAP demonstrated by fluorescence resonance energy transfer microscopy and decreased leukotriene production in urine. CONCLUSION: 5-LO and FLAP colocalize in the interstitium of the renal medulla following T/HS. MLD attenuates this phenomenon, which coincides with pathologic changes seen in tubules during kidney injury and biochemical evidence of AKI. These data suggest that gut-derived leukotriene substrate predisposes the kidney and the lung to subsequent injury.