Porcine Packed Red Blood Cells Demonstrate a Distinct Red Blood Cell Storage Lesion.

INTRODUCTION: The red blood cell (RBC) storage lesion has been well described in mouse and human blood but not in swine. Understanding the porcine RBC storage lesion is necessary prior to evaluating transfusion of stored packed red blood cells (pRBCs) in polytrauma models. We hypothesized that porcine pRBCs would undergo a similar storage lesion severity after 42 d. METHODS: Whole blood was collected from female Yorkshire pigs and pRBCs were isolated in additive storage solution 3. Female human whole blood was obtained from our local blood bank and pRBCs prepared. Human and porcine pRBCs were stored for 42 d and sampled weekly and evaluated for markers of the RBC storage lesion including biochemical measurements, eryptotic RBCs, band-3 expression, erythrocyte-derived microvesicles, and free hemoglobin concentrations. RESULTS: Porcine pRBCs demonstrated a hematocrit similar to human pRBCs. Both human and porcine pRBC units developed a progressive storage lesion. However, over 42 d of storage, porcine pRBCs maintained their pH and had decreased glucose utilization. Porcine pRBCs also demonstrated decreased levels of eryptosis compared to human samples and generated less erythrocyte-derived microvesicles with lower free hemoglobin concentrations. CONCLUSIONS: Porcine pRBCs stored in additive storage solution 3 demonstrate a progressive RBC storage lesion over 42 d of storage but with less severity than human controls. Given the differences in porcine erythrocyte metabolism, further study of the storage lesion in porcine blood is needed in addition to incorporating the use of stored porcine pRBCs in a swine model of hemorrhagic shock to more closely mimic clinical scenarios.

Surviving septic patients endotyped with a functional assay demonstrate active immune responses.

Introduction: Sepsis is a complex clinical syndrome characterized by a heterogenous host immune response. Historically, static protein and transcriptomic metrics have been employed to describe the underlying biology. Here, we tested the hypothesis that ex vivo functional TNF expression as well as an immunologic endotype based on both IFNγ and TNF expression could be used to model clinical outcomes in sepsis patients. Methods: This prospective, observational study of patient samples collected from the SPIES consortium included patients at five health systems enrolled over 17 months, with 46 healthy control patients, 68 ICU patients without sepsis, and 107 ICU patients with sepsis. Whole blood was collected on day 1, 4, and 7 of ICU admission. Outcomes included in-hospital and 180-day mortality and non-favorable discharge disposition defined by skilled nursing facility, long-term acute care facility, or hospice. Whole blood ELISpot assays were conducted to quantify TNF expression [stimulated by lipopolysaccharide (LPS)] and IFNγ expression (stimulated by anti-CD3/CD28 mAb), which were then used for assignment to one of four subgroups including an 'immunocompetent', 'immunosuppressed endotype', and two 'mixed' endotypes. Results: Whole blood TNF spot-forming units were significantly increased in septic and CINS patients on days 4 and 7 compared to healthy subjects. In contrast, TNF expression per cell on days 1, 4, and 7 was significantly lower in both septic and critically ill non-septic (CINS) patients compared to healthy subjects. Early increases in total TNF expression were associated with favorable discharge disposition and lower in-hospital mortality. 'Immunocompetent' endotype patients on day 1 had a higher proportion of favorable to non-favorable discharges compared to the 'immunosuppressed' endotype. Similarly, 'immunocompetent' endotype patients on day 4 had a higher in-hospital survival compared to the 'immunosuppressed' endotype patients. Finally, among septic patients, decreased total TNF and IFNγ expression were associated with 180-day mortality. Conclusions: Increased ex vivo whole blood TNF expression is associated with improved clinical outcomes. Further, the early 'immunocompetent' endotype is associated with favorable discharge and improved in-hospital and 180-day survival. The ability to functionally stratify septic patients based on blood cell function ex vivo may allow for identification of future immune modulating therapies.

Age above all: The impact of storage duration in mice resuscitated with whole blood or packed red blood cells and plasma in a hemorrhagic shock model.

BACKGROUND: The use of whole blood compared with a balanced ratio of components in trauma resuscitation remains an area of ongoing investigation. One factor that may affect outcomes is the age of the blood product transfused. We used a murine model of blood banking and hemorrhagic shock resuscitation to compare the effect of storage duration in whole blood and packed red blood cells on the recipient inflammatory response. METHODS: Murine whole blood or packed red blood cells were evaluated for the red blood cells storage lesion up to 14 days. Mice underwent hemorrhagic shock followed by resuscitation with whole blood or packed red blood cells combined with equal volume of thawed plasma (1:1) stored for 1, 7, or 14 days. Serum and lung cytokine/chemokine levels were measured and leukocyte infiltration determined via immunohistochemistry. RESULTS: Both whole blood and packed red blood cells develop a blood storage lesion. Four hours after resuscitation, mice resuscitated with either day 14 whole blood or 1:1 demonstrated increased inflammatory cytokines and chemokines with similar findings within lung tissue compared with mice resuscitated with whole blood and 1:1 products stored for 1 or 7 days. CONCLUSIONS: Resuscitation with murine packed red blood cells or whole blood stored for 14 days produces a pronounced recipient inflammatory response compared with those units stored for lesser durations. Given the shorter storage duration of human whole blood to packed RBCs, resuscitation with whole blood within current storage limits may represent an advantageous resuscitation strategy compared with older packed red blood cells.

External Validation of Predictors of Mortality in Polytrauma Patients.

INTRODUCTION: The Parkland Trauma Index of Mortality (PTIM) is an integrated, machine learning 72-h mortality prediction model that automatically extracts and analyzes demographic, laboratory, and physiological data in polytrauma patients. We hypothesized that this validated model would perform equally as well at another level 1 trauma center. METHODS: A retrospective cohort study was performed including ∼5000 adult level 1 trauma activation patients from January 2022 to September 2023. Demographics, physiologic and laboratory values were collected. First, a test set of models using PTIM clinical variables (CVs) was used as external validation, named PTIM+. Then, multiple novel mortality prediction models were developed considering all CVs designated as the Cincinnati Trauma Index of Mortality (CTIM). The statistical performance of the models was then compared. RESULTS: PTIM CVs were found to have similar predictive performance within the PTIM + external validation model. The highest correlating CVs used in CTIM overlapped considerably with those of the PTIM, and performance was comparable between models. Specifically, for prediction of mortality within 48 h (CTIM versus PTIM): positive prediction value was 35.6% versus 32.5%, negative prediction value was 99.6% versus 99.3%, sensitivity was 81.0% versus 82.5%, specificity was 97.3% versus 93.6%, and area under the curve was 0.98 versus 0.94. CONCLUSIONS: This external cohort study suggests that the variables initially identified via PTIM retain their predictive ability and are accessible in a different level 1 trauma center. This work shows that a trauma center may be able to operationalize an effective predictive model without undertaking a repeated time and resource intensive process of full variable selection.

Tranexamic Acid Administration Does Not Alter Inflammation After Traumatic Brain Injury, Regardless of Timing.

INTRODUCTION: Tranexamic acid (TXA) administered early after traumatic brain injury (TBI) can decrease morbidity and mortality. The purpose of this study is to determine if the timing of TXA administration after TBI affects postinjury inflammatory markers or phosphorylated tau (p-tau) levels within the hippocampus. METHODS: Male mice (9-11 wk) were split into six groups based on injury and timing of TXA administration (n = 5 per group): Sham, TBI-only, 100 mg/kg TXA-only, TBI + TXA 10 min, TBI + TXA 1 h, and TBI + TXA 6 h. Moderate concussive TBI was induced via weight drop. Serum and brain homogenates were collected at 6 and 24 h postinjury and analyzed for 14 inflammatory cytokines via multiplex enzyme-linked immunosorbent assay. Serum was analyzed for glial fibrillary acidic protein levels. Additional cohorts were survived to 30 d for hippocampal p-tau quantification using immunohistochemistry. RESULTS: Serum levels of interleukin (IL) 1ß (IL-1ß), IL-3, IL-12, IL-17, monocyte chemoattractant protein-1, granulocyte-macrophage colony-stimulating factor, and regulated on activation, normal T-cell expressed and secreted were elevated in TBI mice compared to sham mice at 24 h. Levels of IL-1ß and monocyte chemoattractant protein-1 were lower in 6-h TXA-treated mice than 1-h TXA-treated mice following TBI. IL-12 and macrophage inflammatory protein-1α levels were decreased in 6-h TXA-treated mice compared to 10-min TXA-treated mice. Administration of TXA at 10 min and 6 h but not 1 h postTBI reduced serum glial fibrillary acidic protein levels compared to TBI-only mice. Hippocampal p-tau accumulation was increased after TBI but not reduced by TXA administration. CONCLUSIONS: Our results demonstrate that neither early nor delayed administration of TXA conveyed significant systemic or cerebral benefit in cytokine levels following TBI. Further research should be conducted to assess blood brain barrier integrity and neurobehavioral recovery following TXA administration postTBI.

Multi-Modal Venous ThromboembolicProphylaxis Aids in Risk Reduction Following Splenectomy in Female and Male Mice.

INTRODUCTION: Splenectomy (SPLN) is associated with elevated risk of venous thromboembolic (VTE) disease. Enoxaparin (ENX) is a low-molecular-weight heparin agent used in VTE chemoprophylaxis. Early aspirin administration ameliorates postSPLN platelet hyperaggregability in male mice. Previous literature has excluded female mice, citing potential effects of estrogen on platelet count and activation as a reason. We hypothesized that multimodal therapy using aspirin and ENX would mitigate postoperative platelet aggregability in mice across sexes. METHODS: Murine models of SPLN included both male and female mice. Treatment groups included placebo gavage, sham laparotomy, SPLN alone, SPLN and aspirin, SPLN and ENX, and SPLN with aspirin and ENX (n = 5 per group). Chemoprophylaxis dosing was initiated before SPLN. Mice were euthanized on post-operative day (POD) 1 or 3; platelet counts were obtained and blood samples were analyzed via electrical impedance aggregometry. RESULTS: Females on POD 3 following SPLN demonstrated increased platelet count compared to female mice with no treatment intervention. Male and female mice demonstrated increased adenosine diphosphate (ADP)-induced platelet aggregability on POD 3 following SPLN compared to the placebo group. Treatment with aspirin and ENX decreased this post-SPLN platelet hyperaggregability in both sexes. Females demonstrated significantly higher ADP-mediated platelet aggregability in placebo, SPLN, and SPLN with aspirin and ENX when compared to males of identical treatment groups on POD 3. CONCLUSIONS: Platelet hyperaggregability following SPLN is mediated primarily by ADP in both males and females, but higher relative aggregability is demonstrated in females. Early administration of dual-agent VTE chemoprophylaxis utilizing aspirin and ENX mitigates this hyperaggregability and may aid in VTE risk reduction across sexes.

Development and optimization of a diluted whole blood ELISpot assay to test immune function.

Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.

Aberrant Oxygen Concentrations Induce Systemic Inflammation in a Murine Model.

INTRODUCTION: Hypoxia is a significant cause of secondary insult in the critically ill trauma or surgical patient. The cause of increased mortality following a brief period of hypoxia is not well understood. The aim of this study is to determine the effect of acute, isolated deviations in oxygen concentration on proinflammatory cytokine release and markers of endothelial stress in a murine model. METHODS: Mice were randomized to either control, hypoxia, or hyperoxia group. The control group was exposed to room air for 60 min, the hyperoxia group was exposed to 70% fraction of inspired oxygen, and the hypoxia group was exposed to 10% fraction of inspired oxygen for 60 min. Whole blood collection was completed via cardiac puncture. Serum concentrations of proinflammatory cytokines and endothelial stress markers were analyzed via enzyme-linked immunosorbent assay. RESULTS: Following exposure to hypoxic conditions, there was a significant increase in interleukin (IL)-1α (IL-1 α), IL-1 ß, IL-3, IL-4, IL-6, IL-10, tumor necrosis factor α . Following exposure to hyperoxic conditions, there was a significant increase in monocyte chemoattractant protein-1 and regulated upon activation normal T cell expressed and presumably secreted, as well as a significant decrease in IL-12, and IL-17. No clinically significant difference was noted in serum concentration of endothelial stress markers between the treatment groups. DISCUSSION: Exposure to oxygen extremes induces systemic inflammation as measured by proinflammatory cytokines in a murine model. Hyperoxia also demonstrates the ability to downregulate certain inflammatory pathways while inducing others. No effect on serum concentration of endothelial stress markers is observed following acute, isolated hypoxic or hyperoxic conditions.

The Next Generation: Surgeon Learning Curve in a Mature Operative Rib Management Program.

INTRODUCTION: Prior work has demonstrated utility in using operative time to measure surgeon learning for surgical stabilization of rib fractures (SSRF); however, no studies have used operative time to evaluate the benefit of proctoring in subsequent generations of surgeons. We sought to evaluate whether there is a difference in learning between an original series (TOS) of self-taught surgeons versus the next generation (TNG) of proctored surgeons using cumulative summation (CUSUM) analysis. We hypothesized that TNG would have a comparatively accelerated learning curve. METHODS: A single-center retrospective review of all SSRF at a level 1 trauma center was performed. Data were collected from the beginning of an operative chest injury program to include at least 2 y of TNG experience. Operative time was used to determine success and misstep based on prior methods. Learning curves using CUSUM analysis were calculated based on an anticipated success rate of 90% and compared between TOS and TNG groups. RESULTS: Over 7 y, 163 patients with a median Injury Severity Score of 24 underwent SSRF. Median operative time was 165 min with a 0.5 plate-to-fracture ratio. All three TOS surgeons experienced a positive slope indicative of early missteps for their first 15-20 cases. By contrast, all three TNG surgeons demonstrated a series of early successes resulting in negative CUSUM slopes which coincided with a period of proctoring. By the end of TNG series, the composite cumulative score was less than half of the TOS surgeon' scores. CONCLUSIONS: Operative time continues to be a useful surrogate for observing SSRF learning curves. In a mature institutional program, proctored novice surgeons appear to have an accelerated learning curve compared to novice surgeons developing a new operative rib program.

Need for Blood Transfusion Volume Is Associated With Increased Mortality in Severe Traumatic Brain Injury.

INTRODUCTION: Many patients suffering from isolated severe traumatic brain injury (sTBI) receive blood transfusion on hospital arrival due to hypotension. We hypothesized that increasing blood transfusions in isolated sTBI patients would be associated with an increase in mortality. METHODS: We performed a trauma quality improvement program (TQIP) (2017-2019) and single-center (2013-2021) database review filtering for patients with isolated sTBI (Abbreviated Injury Scale head ≥3 and all other areas ≤2). Age, initial Glasgow Coma Score (GCS), Injury Severity Score (ISS), initial systolic blood pressure (SBP), mechanism (blunt/penetrating), packed red blood cells (pRBCs) and fresh frozen plasma (FFP) transfusion volume (units) within the first 4 h, FFP/pRBC ratio (4h), and in-hospital mortality were obtained from the TQIP Public User Files. RESULTS: In the TQIP database, 9257 patients had isolated sTBI and received pRBC transfusion within the first 4 h. The mortality rate within this group was 47.3%. The increase in mortality associated with the first unit of pRBCs was 20%, then increasing approximately 4% per unit transfused to a maximum mortality of 74% for 11 or more units. When adjusted for age, initial GCS, ISS, initial SBP, and mechanism, pRBC volume (1.09 [1.08-1.10], FFP volume (1.08 [1.07-1.09]), and FFP/pRBC ratio (1.18 [1.08-1.28]) were associated with in-hospital mortality. Our single-center study yielded 138 patients with isolated sTBI who received pRBC transfusion. These patients experienced a 60.1% in-hospital mortality rate. Logistic regression corrected for age, initial GCS, ISS, initial SBP, and mechanism demonstrated no significant association between pRBC transfusion volume (1.14 [0.81-1.61]), FFP transfusion volume (1.29 [0.91-1.82]), or FFP/pRBC ratio (6.42 [0.25-164.89]) and in-hospital mortality. CONCLUSIONS: Patients suffering from isolated sTBI have a higher rate of mortality with increasing amount of pRBC or FFP transfusion within the first 4 h of arrival.

Platelet Function is Independent of Sphingolipid Manipulation.

INTRODUCTION: Previous literature suggests that sphingolipids may impact systemic coagulation and platelet aggregation, thus modulating the risks of thrombotic events. The goal of this investigation was to evaluate the role of serum sphingolipids on intrinsic platelet function to assess whether pharmacologic manipulation of sphingolipid metabolites would impact platelet aggregability. METHODS: C57BL/6J mice were injected with either normal saline, 1 mg/kg FTY720 (synthetic sphingosine-1-phosphate [S1P] receptor analog), or 5 mg/kg SLM6031434 (sphingosine kinase two inhibitor). Mice were sacrificed at 6 h and whole blood (WB) was collected for impedance aggregometry assessing platelet responsiveness to arachidonic acid or adenosine diphosphate. Ex vivo studies utilized WB or platelet-rich plasma that was pretreated with S1P, FTY720, amitriptyline, or d-sphingosine then analyzed by aggregability and flow cytometry for platelet and platelet-derived microvesicle characteristics. RESULTS: FTY720 and SLM6031434 pretreated induced similar arachidonic acid and adenosine diphosphate-mediated platelet aggregation as controls. Ex vivo WB and platelet-rich plasma treatment with S1P, FTY720, amitriptyline and d-sphingosine did not impact platelet aggregation. The percentages of CD41+, CD62P+ and CD41+/ceramide+, CD62P+/ceramide + platelets, and platelet-derived microvesicle were not significantly different between amitriptyline-treated and normal saline-treated cohorts. CONCLUSIONS: Sphingolipid modulating agents, such as FTY720, SLM6031434, S1P, amitriptyline, ceramide, and d-sphingosine do not appear to independently impact platelet aggregation in murine models.

Re-evaluating the Use of High Sensitivity Troponin to Diagnose Blunt Cardiac Injury.

INTRODUCTION: Blunt cardiac injury (BCI) can be challenging diagnostically, and if misdiagnosed, can lead to life-threatening complications. Our institution previously evaluated BCI screening with troponin and electrocardiogram (EKG) during a transition from troponin I to high sensitivity troponin (hsTnI), a more sensitive troponin I assay. The previous study found an hsTnI of 76 ng/L had the highest capability of accurately diagnosing a clinically significant BCI. The aim of this study was to determine the efficacy of the newly implemented protocol. METHODS: Patients diagnosed with a sternal fracture from March 2022 to April 2023 at our urban level-1 trauma center were retrospectively reviewed for EKG findings, hsTnI trend, echocardiogram changes, and clinical outcomes. The BCI cohort and non-BCI cohort ordinal measures were compared using Wilcoxon's two-tailed rank sum test and categorical measures were compared with Fisher's exact test. Youden indices were used to evaluate hsTnI sensitivity and specificity. RESULTS: Sternal fractures were identified in 206 patients, of which 183 underwent BCI screening. Of those screened, 103 underwent echocardiogram, 28 were diagnosed with clinically significant BCIs, and 15 received intervention. The peak hsTnI threshold of 76 ng/L was found to have a Youden index of 0.31. Rather, the Youden index was highest at 0.50 at 40 ng/L (sensitivity 0.79 and specificity 0.71) for clinically significant BCI. CONCLUSIONS: Screening patients with sternal fractures for BCI using hsTnI and EKG remains effective. To optimize the hsTnI threshold, this study determined the hsTnI threshold should be lowered to 40 ng/L. Further improvements to the institutional protocol may be derived from multicenter analysis.

Systemic Inflammatory Effect of Hypobaria During Aeromedical Evacuation after Porcine Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI)-related morbidity is caused largely by secondary injury resulting from hypoxia, excessive sympathetic drive, and uncontrolled inflammation. Aeromedical evacuation (AE) is used by the military for transport of wounded soldiers to higher levels of care. We hypothesized that the hypobaric, hypoxic conditions of AE may exacerbate uncontrolled inflammation after TBI that could contribute to more severe TBI-related secondary injury. STUDY DESIGN: Thirty-six female pigs were used to test TBI vs Sham TBI, hypoxia vs normoxia, and hypobaria vs ground conditions. TBI was induced by controlled cortical injury, hypobaric conditions of 12,000 ft were established in an altitude chamber, and hypoxic exposure was titrated to 85% SpO 2 while at altitude. Serum cytokines, ubiquitin C-terminal hydrolase L1, and TBI biomarkers were analyzed via ELISA. Gross analysis and staining of cortex and hippocampus tissue was completed for glial fibrillary acidic protein and phosphorylated tau. RESULTS: Serum interleukin-1ß, interleukin-6, and tumor necrosis factor-α were significantly elevated after TBI in pigs exposed to altitude-induced hypobaria/hypoxia, as well as hypobaria alone, compared with ground level/normoxia. No difference in TBI biomarkers after TBI or hypobaric, hypoxic exposure was noted. No difference in brain tissue glial fibrillary acidic protein or phosphorylated tau when comparing the most different conditions of Sham TBI + ground or normoxia with the TBI + hypobaria/hypoxia group was noted. CONCLUSIONS: The hypobaric environment of AE induces systemic inflammation after TBI. Severe inflammation may play a role in exacerbating secondary injury associated with TBI and contribute to worse neurocognitive outcomes. Measures should be taken to minimize barometric and oxygenation changes during AE after TBI.

Direct red blood cell effect on thrombosis is dependent on the interaction of tissue factor and calcium with membrane phosphatidylserine.

BACKGROUND: Prior literature has implicated red blood cells (RBCs) in the initiation of thrombosis and suggests that posttransfusion hypercoagulability may occur secondary to the effects of RBCs. Elevated serum tissue factor is a known sequelae of acute trauma. Phosphatidylserine (PS) is a prothrombotic phospholipid present within the RBC cell membrane. We hypothesized that RBC aggregation is dependent on the interaction between RBC membrane bound (exposed) PS, extracellular calcium, and tissue factor. METHODS: Human whole blood (WB) was separated into components, including RBCs and platelet-rich plasma (PRP). Whole blood, PRP, and RBCs underwent impedance aggregometry utilizing arachidonic acid (AA), ADP, collagen, calcium, and tissue factor (TF)-based agonists. Red blood cells then underwent impedance aggregometry utilizing combined calcium and TF agonists. Red blood cells were pretreated with Annexin V, a known PS blocking agent, and underwent impedance aggregometry with combined calcium and TF agonists to determine if the mechanism of calcium/TF-induced RBC aggregability is dependent on PS. Red blood cells treated with calcium, TF, calcium+TF, and pre-treated with Annexin V followed by calcium+TF were perfused through an in vitro model of pulmonary microcirculatory flow. RESULTS: Red blood cell aggregation was significantly higher than that of WB and PRP when utilizing a TF agonist, an effect unique to TF. The combination of calcium and TF demonstrated significantly higher RBC aggregation than either agonist alone. Pretreatment with Annexin V resulted in a significantly reduced aggregability of RBC following treatment with TF + calcium. Red blood cells aged to 42 days did not exhibit significant change in aggregation. Exposure to calcium and TF significantly reduced time to thrombosis of RBCs perfused through a pulmonary microcirculatory model. CONCLUSION: Treatment with both TF and calcium synergistically induces RBC aggregation. Phosphatidylserine appears to play an integral role in the TF/calcium-based, age-independent RBC aggregation response. Red blood cells treated with TF + calcium exhibit more rapid thrombus formation in an in vitro model of pulmonary microcirculatory perfusion.

Desmopressin, Misoprostol, nor Carboprost Affect Platelet Aggregability Following Traumatic Brain Injury and Aspirin.

INTRODUCTION: Desmopressin (DDAVP) has been utilized clinically in patients taking aspirin (ASA) to improve drug-induced platelet dysfunction. Misoprostol and carboprost, prostaglandin analogs commonly used for postpartum hemorrhage, may also induce platelet aggregation. The aim of this study was to determine the effects of DDAVP, misoprostol, and carboprost administration on platelet aggregability following traumatic brain injury (TBI) in mice treated with ASA. METHODS: Male C57BL/6 mice were randomized into seven groups (n = 5 each): untouched, ASA only, Saline/TBI, ASA/TBI, ASA/TBI/DDAVP 0.4 µg/kg, ASA/TBI/misoprostol 1 mg/kg, and ASA/TBI/carboprost 100 µg/kg. TBI was induced via a weight drop model 4-h after ASA (50 mg/kg) gavage. Mice were given an intraperitoneal injection of DDAVP, misoprostol, or carboprost 10 minutes after TBI. In vivo testing was completed utilizing tail vein bleed. Mice were sacrificed 30-min posttreatment and blood was collected via cardiac puncture. Whole blood was analyzed via Multiplate impedance aggregometry, rotational thromboelastometry, and TEG6s. RESULTS: Mice receiving misoprostol after ASA/TBI demonstrated decreased tail vein bleeding times compared to ASA only treated mice. However, mice treated with misoprostol following ASA and TBI demonstrated decreased platelet aggregability compared to untouched mice and TBI only mice within the arachidonic acid agonist pathway. By contrast, DDAVP and carboprost did not significantly change platelet aggregability via adenosine diphosphate or arachidonic acid following ASA and TBI. However, DDAVP did decrease the platelet contribution to clot via rotational thromboelastometry. CONCLUSIONS: Reversal of medication-induced platelet inhibition has become increasingly controversial after TBI. Based on these results, DDAVP, misoprostol, nor carboprost consistently improve platelet aggregability following TBI in those also treated with ASA.

Just Say NO: Inhaled Nitric Oxide Effect on Respiratory Parameters Following Traumatic Brain Injury in Humans and a Porcine Model.

INTRODUCTION: The mechanism of post-traumatic brain injury (TBI) hypoxemia involves ventilation/perfusion mismatch and loss of pulmonary hypoxic vasoconstriction. Inhaled nitric oxide (iNO) has been studied as an adjunct treatment to avoid the use of high positive end-expiratory pressure and inspired oxygen in treatment-refractory hypoxia. We hypothesized that iNO treatment following TBI would improve systemic and cerebral oxygenation via improved matching of pulmonary perfusion and ventilation. METHODS: Thirteen human patients with isolated TBI were enrolled and randomized to receive either placebo or iNO with measured outcomes including pulmonary parameters, blood gas data, and intracranial pressure (ICP) /perfusion. To complement this study, a porcine model of TBI (including 10 swine) was utilized with measured outcomes of brain tissue blood flow and oxygenation, ventilator parameters, and blood gas data both after administration and following drug removal and clearance. RESULTS: There were no clinically significant changes in pulmonary parameters in either the human or porcine arm following administration of iNO when compared to either the placebo group (human arm) or the internal control (porcine arm). Analysis of pooled human data demonstrated the preservation of alveolar recruitment in TBI patients. There were no clinically significant changes in human ICP or cerebral perfusion pressure following iNO administration compared to controls. CONCLUSIONS: iNO had no significant effect on clinically relevant pulmonary parameters or ICPs following TBI in both human patients and a porcine model. The pressure-based recruitment of the human lungs following TBI was preserved. Further investigation will be needed to determine the degree of utility of iNO in the setting of hypoxia after polytrauma.

Microvesicles from stored red blood cells induce P-selectin and von Willebrand factor release from endothelial cells via a protein kinase C-dependent mechanism.

INTRODUCTION: The use of packed red blood cells (pRBCs) for resuscitation is limited by the red blood cell storage lesion, a series of biochemical and physiological changes that occur during the storage and aging of blood. Microvesicles (MVs) shed from pRBCs during this process are one component of the red blood cell storage lesion and lead to acute lung injury and pulmonary vascular microthrombi. We hypothesized that MVs from stored pRBCs lead to the release of P-selectin and von Willebrand factor (vWF) from endothelial cells and that this mechanism is mediated via activation of protein kinase C (PKC) or protein kinase A (PKA). METHODS: Leukoreduced, platelet-poor murine pRBCs were isolated from C57BL/6 8-12 week-old male mice via cardiac puncture, prepared via centrifugation using a Ficoll gradient, and stored for up to 14 days, the equivalent of 42 days of storage in humans. MVs were isolated from the stored pRBC units via sequential high-speed centrifugation. Murine lung endothelial cells (MLECs) were cultured and grown to confluence, then treated with MVs and either calphostin C, a PKC inhibitor (10 µg/mL), or PKI 14-22 amide, a PKA inhibitor (10 µM). The supernatant was collected after 1 h. P-selectin and vWF A2 concentrations were quantified via ELISA. Immunofluorescent staining for vWF was performed on MLECs. Statistical analysis was performed via unpaired t-test or ANOVA as indicated and reported as mean ± SD. Concentration is reported as pg/mL. RESULTS: MLECs treated with MVs isolated from stored pRBCs demonstrated increased release of P-selectin and vWF A2 in a dose-dependent fashion. MLECs treated with MVs prepared from stored as compared to fresh pRBCs demonstrated increased release of P-selectin (3751 ± 726 vs 359 ± 64 pg/mL, p < 0.0001) and vWF A2 (3141 ± 355 vs 977 ± 75 pg/mL, p < 0.0001) with increasing duration of storage. The treatment of MVs with calphostin C decreased the amount of P-selectin (1471 ± 444 vs 3751 ± 726 pg/mL, p < 0.0001) and VWF A2 (2401 ± 289 vs 3141 ± 355 pg/mL, p = 0.0017) released into the supernatant by MLECs compared to MVs alone. The treatment of MVs with PKI 14-22 increased the amount of P-selectin released compared to MVs alone (1999 ± 67 vs 1601 ± 135 pg/mL, p = 0.0018). CONCLUSIONS: MVs from stored pRBCs stimulate the release of P-selectin and VWF A2 from endothelial cells. The effect of MVs increases with both dose of MVs and age of stored pRBCs from which they are formed. This mechanism is dependent on activation of PKC and inhibition of this enzyme represents a potentially significant strategy to modulate the inflammatory response to resuscitation with stored pRBCs.

Murine Traumatic Brain Injury Model Comparison: Closed Head Injury Versus Controlled Cortical Impact.

INTRODUCTION: Various murine models have been utilized to study TBI, including closed head injury (CHI) and controlled cortical impact (CCI), without direct comparison. The aim of our study was to evaluate these models to determine differences in neurological and behavioral outcomes postinjury. METHODS: Male C57B/6 mice (9-10 wk) were separated into six groups including: untouched, sham craniotomy (4 mm), CCI 0.9 mm depth of impact, CCI 1.6 mm, CCI 2.2 mm, and CHI. CCI was performed using a 3 mm impact tip at a velocity of 5 m/s, dwell time of 250 ms, and depth as noted above. CHI was completed with a centered 400 g weight drop from 1 cm height. Mice were survived to 14-d (n = 5 per group) and 30-d (n = 5 per group) respectively for histological analysis of p-tau within the hippocampus. These mice underwent Morris Water Maze memory testing and Rotarod motor testing. Serum was collected from a separate cohort of mice (n = 5 per group) including untouched, isoflurane only, CCI 1.6 mm, CHI at 1, 4, 6, and 24 h for analysis of neuron specific enolase and glial fibrillary acidic protein (GFAP) via ELISA. Laser speckle contrast imaging was analyzed prior to and after impact in the CHI and CCI 1.6 mm groups. RESULTS: There were no significant differences in Morris Water Maze or Rotarod testing times between groups at 14- or 30-d. P-tau was significantly elevated in all groups except CCI 1.6 mm contralateral and CCI 2.2 mm ipsilateral compared to untouched mice at 30-d. P-tau was also significantly elevated in the CHI group at 30 d compared to CCI 1.6 mm contralateral and CCI 2.2 mm on both sides. GFAP was significantly increased in mice undergoing CHI (9959 ± 91 pg/mL) compared to CCI (2299 ± 1288 pg/mL), isoflurane only (133 ± 75 pg/mL), and sham (86 ± 58 pg/mL) at 1-h post TBI (P < 0.0001). There were no differences in serum neuron specific enolase levels between groups. Laser doppler imaging demonstrated similar decreases in cerebral blood flow between CHI and CCI; however, CCI mice had a reduction in blood flow with craniotomy only that did not significantly decrease further with impact. CONCLUSIONS: Based on our findings, CHI leads to increased serum GFAP levels and increased p-tau within the hippocampus at 30-d postinjury. While CCI allows the comparison of one cerebral hemisphere to the other, CHI may be a better model of TBI as it requires less technical expertise and has similar neurological outcomes in these murine models.

Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot.

BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.

Syndecan-1 as the Effect or Effector of the Endothelial Inflammatory Response?

INTRODUCTION: Syndecan-1 is a heparan sulfate proteoglycan found in the glycocalyx of vascular endothelial cells. Serum levels of syndecan-1 have repeatedly been demonstrated to increase following traumatic injury and shock, but it is unclear whether syndecan-1 plays an active role in the inflammatory response or is simply a biomarker of a state of hypoperfusion. The aim of this study was to identify the role of syndecan-1 role in the inflammatory process in the absence of trauma. METHODS: Male mice were randomized into five groups (n = 3). Four groups received increasing concentrations of syndecan-1 (1, 10, 100, and 1000pg/mL per blood volume) and a fifth group was given normal saline as a control via intravenous injection. These concentrations were selected based on previous syndecan-1 enzyme-linked immunosorbent assay data acquired following induced hemorrhagic shock in mice resulting in serum levels of 10-6000 pg/mL. Mice from each group were sacrificed at 1-, 4-, and 24-h time points for serum biomarker evaluation. A multiplex enzyme-linked immunosorbent assay was performed to analyze proinflammatory cytokines and chemokines including interleukin (IL)-1a, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-17, monocyte chemoattractant protein-1, TNF-α, macrophage inflammatory protein-1α, granulocyte-macrophage colony-stimulating factor, and normal T cell expressed and presumably secreted levels. Whole blood was analyzed via rotational thromboelastometry in a separate group of mice dosed with syndecan-1 at 1000 pg/mL and compared to sham mice at 1 h. RESULTS: Tumor necrosis factor-α was significantly elevated in the 1000 pg/mL group compared to sham animals. There were no significant changes in IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, monocyte chemoattractant protein--1, macrophage inflammatory protein-1α, granulocyte-macrophage colony-stimulating factor, or normal T cell expressed and presumably secretedat 1, 4, and 24 h for any group when compared to mice receiving saline alone. No significant differences were noted in coagulability between the 1000 pg/mL syndecan-1 group and shams at 1 h CONCLUSIONS: Inflammatory cytokine concentrations did not change with increasing dosage of syndecan-1 within mice at any timepoint, except for an acute change in tumor necrosis factor-α which was transient. Based on our results, syndecan-1 appears to be a biomarker for inflammation rather than an active participant in eliciting an inflammatory response. Further research will focus on the role of syndecan-1 following hemorrhagic shock.