Autologous bone marrow mononuclear cells to treat severe traumatic brain injury in children.

Autologous bone marrow mononuclear cells (BMMNCs) infused after severe traumatic brain injury have shown promise for treating the injury. We evaluated their impact in children, particularly their hypothesized ability to preserve the blood-brain barrier and diminish neuroinflammation, leading to structural CNS preservation with improved outcomes. We performed a randomized, double-blind, placebo-sham-controlled Bayesian dose-escalation clinical trial at two children's hospitals in Houston, TX and Phoenix, AZ, USA (NCT01851083). Patients 5-17 years of age with severe traumatic brain injury (Glasgow Coma Scale score ≤ 8) were randomized to BMMNC or placebo (3:2). Bone marrow harvest, cell isolation and infusion were completed by 48 h post-injury. A Bayesian continuous reassessment method was used with cohorts of size 3 in the BMMNC group to choose the safest between two doses. Primary end points were quantitative brain volumes using MRI and microstructural integrity of the corpus callosum (diffusivity and oedema measurements) at 6 months and 12 months. Long-term functional outcomes and ventilator days, intracranial pressure monitoring days, intensive care unit days and therapeutic intensity measures were compared between groups. Forty-seven patients were randomized, with 37 completing 1-year follow-up (23 BMMNC, 14 placebo). BMMNC treatment was associated with an almost 3-day (23%) reduction in ventilator days, 1-day (16%) reduction in intracranial pressure monitoring days and 3-day (14%) reduction in intensive care unit (ICU) days. White matter volume at 1 year in the BMMNC group was significantly preserved compared to placebo [decrease of 19 891 versus 40 491, respectively; mean difference of -20 600, 95% confidence interval (CI): -35 868 to -5332; P = 0.01], and the number of corpus callosum streamlines was reduced more in placebo than BMMNC, supporting evidence of preserved corpus callosum connectivity in the treated groups (-431 streamlines placebo versus -37 streamlines BMMNC; mean difference of -394, 95% CI: -803 to 15; P = 0.055), but this did not reach statistical significance due to high variability. We conclude that autologous BMMNC infusion in children within 48 h after severe traumatic brain injury is safe and feasible. Our data show that BMMNC infusion led to: (i) shorter intensive care duration and decreased ICU intensity; (ii) white matter structural preservation; and (iii) enhanced corpus callosum connectivity and improved microstructural metrics.

Low molecular weight heparin decreases pro-coagulant activity in clinical MSC products.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are multipotent adult cells that can be isolated from tissues including bone marrow [MSC(BM)], adipose [MSC(AT)] and umbilical cord [MSC(CT)]. Previous studies have linked expression of tissue factor (TF) on MSC surfaces to a procoagulant effect. Venous thromboembolism (VTE), immediate blood-mediated inflammatory reaction (IBMIR) and microvascular thrombosis remain a risk with intravascular MSC therapy. We examined the effect of low molecular weight heparin (LMWH) on clinical-grade MSCs using calibrated automated thrombography (CAT). METHODS: Clinical grade MSC(BM)s, MSC(AT)s and MSC(CT)s harvested at passage 4 were added to normal pooled plasma (NPP) to a final concentration of either 400 000 or 50 000 cells/mL. LMWH was added to plasma in increments of 0.1 U/mL. Thrombin generation (TG) was measured using CAT. Flow cytometry was conducted on the cells to measure MSC phenotype and TF load. RESULTS: Presence of MSCs decreased lag time and increased peak TG. All cell lines demonstrated a dose response to LMWH, with MSC(AT) demonstrating the least thrombogenicity and most sensitivity to LMWH. TG was significantly reduced in all cell lines at doses of 0.2 U/mL LMWH and higher. DISCUSSION: All MSC types and concentrations had a decrease in peak thrombin and TG with increasing amounts of LMWH. While this in vitro study cannot determine optimal dosing, it suggests that LMWH can be effectively used to lower the risk of VTE associated with intravascular administration of MSCs. Future in vivo work can be done to determine optimal dosing and effect on IBMIR and VTE.

Cytokine Release by Microglia Exposed to Neurologic Injury Is Amplified by Lipopolysaccharide.

INTRODUCTION: Traumatic brain injury (TBI) is a leading cause of death and morbidity in the trauma population. Microglia drive the secondary neuroinflammatory response after TBI. We sought to determine if the microglial response to neurologic injury was exacerbated by a second stimulus after exposure to neurologic injury. METHODS: Sprague-Dawley rats (age 2-3 wk) were divided into injured and noninjured groups. Injured rats underwent a controlled cortical impact injury; noninjured rats remained naïve to any injury and served as the control group. Primary rat microglia were isolated and applied to in vitro cultures. After incubation for 24 h, the microglia were stimulated with lipopolysaccharide (LPS) or norepinephrine. Twenty-four hours after stimulation, cell culture supernatant was collected. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) production were measured by standard enzyme-linked immunosorbent assays. GraphPad Prism was used for statistical analysis. RESULTS: When compared to noninjured microglia, LPS induced a significantly greater production of TNF-α in microglia isolated from the injured ipsilateral (versus noninjured = 938.8 ± 155.1, P < 0.0001) and injured contralateral hemispheres (versus noninjured = 426.6 ± 155.1, P < 0.0001). When compared to microglia from noninjured cerebral tissue, IL-6 production was significantly greater after LPS stimulation in the injured ipsilateral hemisphere (mean difference versus noninjured = 9540 ± 3016, P = 0.0101) and the contralateral hemisphere (16,700 ± 3016, P < 0.0001). Norepinephrine did not have a significant effect on IL-6 or TNF-α production. CONCLUSIONS: LPS stimulation may amplify the release of proinflammatory cytokines from postinjury microglia. These data suggest that post-TBI complications, like sepsis, may propagate neuroinflammation by augmenting the proinflammatory response of microglia.

Risk Factors for Blunt Cerebrovascular Injury in a Cohort of Pediatric Patients With Cervical Seat Belt Sign.

BACKGROUND: Blunt cerebrovascular injury (BVCI), injury to the carotid or vertebral arteries, may result from forces involving seatbelts. Although previous studies have not found a seat belt sign to be a significant predictor for BCVI, it is still used to screen patients for BCVI. OBJECTIVE: This study aims to determine risk factors for BCVI within a cohort of patients with seat belt signs. METHODS: We conducted a retrospective cohort study using our institutional trauma registry and included patients younger than 18 years with blunt trauma who both had a computed tomography angiography (CTA) of the neck performed and had evidence of a seat belt sign per the medical record. We reported frequencies, proportions, and measures of central tendency and conducted univariate analysis to evaluate factors associated with BCVI. We estimated the magnitude of the effect of each variable associated with the study outcome by conducting logistic regression and reporting odds ratios and 95% confidence intervals. RESULTS: Among all study patients, BCVI injuries were associated with Injury Severity Score higher than 15 ( P = 0.04), cervical spinal fractures ( P = 0.007), or basilar skull fractures ( P = 0.01). We observed higher proportions of children with BCVI when other motorized and other blunt mechanisms were reported as the mechanisms of injury ( P = 0.002) versus motor vehicle collision. CONCLUSIONS: Significant risk factors for BCVI in the presence of seat belt sign are: Injury severity score greater than 15, cervical spinal fracture, basilar skull fracture, and the other motorized mechanism of injury, similar to those in all children at risk of BCVI.

A decade of blood-brain barrier permeability assays: Revisiting old traumatic brain injury rat data for new insights and experimental design.

Increased microvascular permeability at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema following traumatic brain injury (TBI). These pathologic conditions compromise the integrity of the neurovascular unit resulting in severe brain dysfunction. To quantify this permeability and assess ionic equillibrium, preclinical researchers have relied on the use of various molecular weight permeable dyes such as Evans Blue that normally cannot enter the brain parenchyma under homeostatic conditions. Evans Blue, the most cited of the molecular weight dyes, has reported reproducibility issues because of harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra associated with the dye. Our laboratory group transitioned to Alexa Fluor 680, a far-red dye with improved sensitivity compared to Evans Blue and thus improved reproducibility to alleviate this issue. To evaluate our reproducibility and increase the rigor of our experimental design, we retrospectively analyzed our controlled cortical impact (CCI) experiments over the past 10 years to evaluate effect size with larger samples and potential sources of variability. All of our BBB permeability experiments were performed with Male, Sprague Dawley rats weighing between 225 and 300 g. Historically, Sprague Dawleys were randomly divided into treatment groups: SHAM, CCI, and a stem cell-based treatment from years 2007-2020. The assessment of microvascular hyperpermeability were evaluated by comparing the mean at minimum threshold, area at 1 k-2 k, and intensity density obtained from Alexa Fluor 680 permeability data. Studies utilizing Evans Blue were further compared by tip depth, diameter size, and the hemisphere of injury. Statistical evaluation utilizing the G Power software analysis did not yield a significant difference in sample size comparing experimental groups for Evans Blue and Alexa Fluor 680 analyzed brain tissue. Our analysis also demonstrated a trend in that recent studies (years 2018-2020) have yielded more compact sample sizes between experimental groups in Alexa Fluor 680 analyzed rats. This retrospective study further revealed that Alexa Fluor 680 image analysis provides greater sensitivity to BBB permeability following TBI in comparison to Evans Blue. Significant differences in sample size were not detected between Evans Blue and Alexa Fluor 680; there were significant differences found throughout year to year analysis at the lower range of thresholds. SUMMARY STATEMENT: This work provides a comparative analysis of BBB permeability assay techniques after CCI model of injury in rats.

Citrate Phosphate Dextrose Alters Coagulation Dynamics Ex Vivo.

INTRODUCTION: Citrate-phosphate-dextrose (CPD) is the most common anticoagulant for blood product storage in the United States. It was developed to prolong shelf life, though there is little research regarding its impact on function following transfusion. We used flow cytometry (FC), thromboelastography (TEG), and a clot contraction assay called the zFlex platform to measure platelet activation and global clot formation in blood samples anticoagulated with either CPD or in a standard blue top citrate (BTC) tube. METHODS: Samples were obtained through venipuncture of the antecubital fossa from healthy donors who had not recently taken antiplatelet medication. Samples for FC analysis were spun to obtain platelet-rich plasma, while TEG and zFlex utilized recalcified whole blood. RESULTS: Mean fluorescence intensity for CD62p (P-selectin, marker of platelet activation) in baseline samples was equal, while mean fluorescence intensity in samples activated with thrombin receptor activating peptide was higher in CPD than BTC (65,814 ± 4445 versus 52,483 ± 5435, P = 0.007). TEG results demonstrated similar maximum amplitude for CPD (62.7 ± 1.8 mm versus 61 ± 1 mm) (P = 0.33), though reaction time and kinetics time were significantly longer in CPD versus BTC. CPD R-time: 7.9 ± 0.4 min versus BTC: 3.8 ± 0.4 (P < 0.001). CPD K-time: 2.2 ± 0.2 min versus BTC: 1.6 ± 0.1 min (P < 0.001). Clot contraction strength was not different between the two groups on zFlex: CPD 4353 ± 6 = 517 µN versus BTC 4901 ± 390 µN (P = 0.39). CONCLUSIONS: Our findings suggest that CPD does not affect platelet function (minimal difference on FC and no difference in ultimate clot strength, which is ∼80% due to platelet function) but may alter clot dynamics by attenuating thrombin generation.

Design and Development of a Clot Burst Pressure Device to Investigate Resuscitation Strategies.

INTRODUCTION: A reduction in clot strength is a hallmark feature of trauma-induced coagulopathy. A better understanding of clot integrity can optimize resuscitation strategies. We designed a device to gauge clot strength by pressurizing fluids over a formed clot and measuring the pressure needed to dislodge the clot. We hypothesized that this device could distinguish between clots formed in hypocoagulable and hypercoagulable states by observing differences in the clot burst pressure. METHODS: Whole blood from healthy volunteers was collected into sodium citrate tubes and was treated with heparin or fibrinogen to generate clots in a hypocoagulable or hypercoagulable state, respectively. Small bore holes were drilled into polystyrene plates, and recalcified blood was pipetted into the holes. Plates were incubated at 37°C for 30 min to form clots. A pressure cap with an inlet for fluid from a syringe pump and an outlet leading to a measurement column was secured in the wells with a watertight seal. RESULTS: Clot burst pressure was normalized to individual baseline values to account for inherent differences in clot strength. The 1.0 g/L and 2.0 g/L fibrinogen groups were 1.65 ± 0.07 (P = 0.0078) and 2.26 ± 0.16 (P = 0.0078) times as strong as baseline, respectively. The 0.10, 0.15, or 0.20 USP units/mL groups were 0.388 ± 0.07 (P = 0.125), 0.31 ± 0.07 (P = 0.125), 0.21 ± 0.07 (P = 0.125) times as strong as baseline, respectively. Data were analyzed using Wilcoxon matched pairs signed rank testing. CONCLUSIONS: This device tests clot strength using burst pressure, an easily interpreted clinical parameter not measured in existing devices. Future work can test blood from trauma patients to better understand trauma pathophysiology.

Risk factors for blunt cerebrovascular injury in the pediatric patient: A systematic review.

BACKGROUND: While blunt cerebrovascular injury (BCVI) is a rare complication of blunt trauma, it is associated with significant morbidity and mortality. In the pediatric population, unique anatomy and development require screening criteria that accurately diagnose these injuries while limiting unwarranted radiation. METHODS: We searched Medline OVID, EMBASE, and Cochrane Library databases for studies that investigated the risk factors of BCVI in individuals younger than 18 years of age. We adhered to the Preferred Reporting Items in Systematic Reviews and Meta-Analyses (PRISMA) guidelines and assessed the quality of each study using the Newcastle-Ottawa Scale. We compared key characteristics of the papers, including incidence of BCVI, incidence of risk factors, and statistical significance of risk factors. RESULTS: Of 1304 studies, 16 met the inclusion criteria. Of these, 15 were retrospective cohort studies and one was a retrospective case control study. Most of the studies included all pediatric blunt trauma admissions, but four only included those which underwent imaging, one only included those with cervical seatbelt sign, and one excluded those who did not survive 24-h post-admission. The ages included as pediatric varied between papers. Papers examined different risk factors and reported differing statistical significances. Though no single risk factor was found to be statistically significant in every study, cervical spine and skull fractures were found to be significant by most. Maxillofacial fractures, depressed GCS score, and stroke were found to be statistically significant by multiple studies. Twelve studies examined cervical soft tissue injury, and none found it to be statistically significant. CONCLUSIONS: The risk factors most found to be statistically significant for BCVI were cervical spine fracture (10/16 studies), skull fracture (9/16), maxillofacial fractures (7/16), depressed GCS score (5/16), and stroke (5/16). There is a need for prospective studies on this topic. LEVEL OF EVIDENCE: Level III, Systematic Review.

Dexmedetomidine Alters the Inflammatory Profile of Rat Microglia In Vitro.

BACKGROUND: Microglia are a primary mediator of the neuroinflammatory response to neurologic injury, such as that in traumatic brain injury. Their response includes changes to their cytokine expression, metabolic profile, and immunophenotype. Dexmedetomidine (DEX) is an α2 adrenergic agonist used as a sedative in critically ill patients, such as those with traumatic brain injury. Given its pharmacologic properties, DEX may alter the phenotype of inflammatory microglia. METHODS: Primary microglia were isolated from Sprague-Dawley rats and cultured. Microglia were activated using multiple mediators: lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), and traumatic brain injury damage-associated molecular patterns (DAMP) from a rat that sustained a prior controlled cortical impact injury. After activation, cultures were treated with DEX. At the 24-h interval, the cell supernatant and cells were collected for the following studies: cytokine expression (tumor necrosis factor-α [TNFα], interleukin-10 [IL-10]) via enzyme-linked immunosorbent assay, 6-phosphofructokinase enzyme activity assay, and immunophenotype profiling with flow cytometry. Cytokine expression and metabolic enzyme activity data were analyzed using two-way analysis of variance. Cell surface marker expression was analyzed using FlowJo software. RESULTS: In LPS-treated cultures, DEX treatment decreased the expression of TNFα from microglia (mean difference = 121.5 ± 15.96 pg/mL; p < 0.0001). Overall, DEX-treated cultures had a lower expression of IL-10 than nontreated cultures (mean difference = 39.33 ± 14.50 pg/mL, p < 0.0001). DEX decreased IL-10 expression in LPS-stimulated microglia (mean difference = 74.93 ± 12.50 pg/mL, p = 0.0039) and Poly I:C-stimulated microglia (mean difference = 23.27 ± 6.405 pg/mL, p = 0.0221). In DAMP-stimulated microglia, DEX decreased the activity of 6-phosphofructokinase (mean difference = 18.79 ± 6.508 units/mL; p = 0.0421). The microglial immunophenotype was altered to varying degrees with different inflammatory stimuli and DEX treatment. CONCLUSIONS: DEX may alter the neuroinflammatory response of microglia. By altering the microglial profile, DEX may affect the progression of neurologic injury.

Combination therapy with Treg and mesenchymal stromal cells enhances potency and attenuation of inflammation after traumatic brain injury compared to monotherapy.

The inflammatory response after traumatic brain injury (TBI) can lead to significant secondary brain injury and chronic inflammation within the central nervous system. Cell therapies, including mesenchymal stromal cells (MSC), have led to improvements in animal models of TBI and are under investigation in human trials. One potential mechanism for the therapeutic potential of MSC is their ability to augment the endogenous response of immune suppressive regulatory T cells (Treg). We have recently shown that infusion of human cord blood Treg decreased chronic microgliosis after TBI and altered the systemic immune response in a rodent model. These cells likely use both overlapping and distinct mechanisms to modulate the immune system; therefore, combining Treg and MSC as a combination therapy may confer therapeutic benefit over either monotherapy. However, investigation of Treg + MSC combination therapy in TBI is lacking. In this study, we compared the ability MSC + Treg combination therapy, as well as MSC and Treg monotherapies, to inhibit the neuroinflammatory response to TBI in vivo and in vitro. Treg + MSC combination therapy demonstrated increased potency to reduce the neuro- and peripheral inflammatory response compared to monotherapy; furthermore, the timing of infusion proved to be a significant variable in the efficacy of both MSC monotherapy and Treg + MSC combination therapy in vivo and in vitro.

The Role of Inflammatory Mediators in the Development of Gastrointestinal Motility Disorders.

Feeding intolerance and the development of ileus is a common complication affecting critically ill, surgical, and trauma patients, resulting in prolonged intensive care unit and hospital stays, increased infectious complications, a higher rate of hospital readmission, and higher medical care costs. Medical treatment for ileus is ineffective and many of the available prokinetic drugs have serious side effects that limit their use. Despite the large number of patients affected and the consequences of ileus, little progress has been made in identifying new drug targets for the treatment of ileus. Inflammatory mediators play a critical role in the development of ileus, but surprisingly little is known about the direct effects of inflammatory mediators on cells of the gastrointestinal tract, and many of the studies are conflicting. Understanding the effects of inflammatory cytokines/chemokines on the development of ileus will facilitate the early identification of patients who will develop ileus and the identification of new drug targets to treat ileus. Thus, herein, we review the published literature concerning the effects of inflammatory mediators on gastrointestinal motility.

Injury Severity, Arrival Physiology, Coagulopathy, and Outcomes Among the Youngest Trauma Patients.

BACKGROUND: Although physiologic differences exist between younger and older children, pediatric trauma analyses are weighted toward older patients. Trauma-induced coagulopathy, determined by rapid thrombelastography (rTEG), is a predictor of outcome in trauma patients, but the significance of rTEG values among very young trauma patients remains unknown. Our objective was to identify the prehospital or physiologic factors, including rTEG values, that were associated with mortality in trauma patients younger than 5 y old. MATERIALS AND METHODS: Patients younger than 5 y old that met the highest-level trauma activation criteria at an academic children's hospital from 2010-2016 were included. Data regarding demographics, pre-hospital management, laboratory values, injury severity, and outcome were queried. Univariate and multivariate analyses were performed comparing survivors and non-survivors. RESULTS: A total of 356 patients were included. 60% were male, and the median age was 3 y (IQR 1-4). Overall mortality was 13% (n = 45); brain injury (91%) and hemorrhage (9%) were the causes of death. Compared to survivors, rTEG values in nonsurvivors showed longer activated clotting time and slower speed of clot formation. Clot strength was also decreased in nonsurvivors. On stepwise regression modeling, rTEG values were not significant predictors of mortality. Admission base deficit, arrival temperature, and head injury severity were identified as independent predictors of mortality. CONCLUSIONS: While rTEG identified coagulopathy in trauma patients < 5 y old, it was not an independent predictor of mortality. Our findings suggest that trauma providers should pay close attention to admission base deficit, arrival temperature, and head injury severity when managing the youngest trauma patients.

Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation.

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.

Adaptation of the hepatic transudation barrier to sinusoidal hypertension.

The role of the hepatic transudation barrier in determining ascites volume and protein content in chronic liver disease is poorly understood. Therefore, the purpose of the present study was to characterize how chronic sinusoidal hypertension impacts hepatic transudation barrier properties and the transudation rate. The suprahepatic inferior vena cava was surgically constricted, and animals were exposed to either short-term (SVH; 2-3 wk) or long-term venous hypertension (LVH; 5-6 wk). Compared with SVH, LVH resulted in lower peritoneal fluid pressure, ascites volume, and ascites protein concentration. The transudation barrier protein reflection coefficient was significantly higher, and the transudation barrier hydraulic conductivity, transudation rate, and transudate-to-lymph protein concentration ratio were significantly lower in LVH animals compared with SVH animals. The sensitivity of transudation rates to acute changes in interstitial fluid pressures was also significantly lower in LVH animals compared with SVH animals. In contrast, there was no detectable difference in hepatic lymph flow rate or sensitivity of lymph flow to acute changes in interstitial fluid pressures between SVH and LVH animals. Taken together, these data suggest that decreased hepatic transudation barrier permeability to fluid and protein and increased reflection coefficient led to a decrease in the hepatic contribution to ascites volume. The present work, to the best of our knowledge, is the first to quantify an anti-ascites adaptation of the hepatic transudation barrier in response to chronic hepatic sinusoidal hypertension.

Intravenous Bone Marrow Mononuclear Cells for Acute Ischemic Stroke: Safety, Feasibility, and Effect Size from a Phase I Clinical Trial.

Cellular therapy is a promising investigational modality to enhance poststroke recovery. We conducted a single-arm, phase I clinical trial to determine the safety and feasibility of intravenous (IV) administration of autologous bone marrow mononuclear cells (MNCs) after acute ischemic stroke (AIS). Patients with moderate severity of AIS underwent bone marrow harvest followed by IV reinfusion of MNCs within 24-72 hours of onset. A target dose of 10 million cells per kilogram was chosen based on preclinical data. Patients were followed up daily during hospitalization and at 1, 3, 6, 12, and 24 months for incidence of adverse events using laboratory, clinical (12 months), and radiological (24 months) parameters. The trial was powered to detect severe adverse events (SAEs) with incidences of at least 10% and planned to enroll 30 patients. Primary outcomes were study-related SAEs and the proportion of patients successfully completing study intervention. A propensity score-based matched control group was used for the estimation of effect size (ES) for day-90 modified Rankin score (mRS). There were no study-related SAEs and, based on a futility analysis, enrolment was stopped after 25 patients. All patients successfully completed study intervention and most received the target dose. Secondary analysis estimated the ES to be a reduction of 1 point (95% confidence interval: 0.33-1.67) in median day-90 mRS for treated patients as compared with the matched control group. Bone marrow harvest and infusion of MNCs is safe and feasible in patients with AIS. The estimated ES is helpful in designing future randomized controlled trials. Stem Cells 2019;37:1481-1491.

A Novel Platelet Function Assay for Trauma.

BACKGROUND: Platelet function tests such as thrombelastography platelet mapping and impedance aggregometry have demonstrated universal platelet dysfunction in trauma patients. In this study, we introduce the measurement of platelet contraction force as a test of platelet function. We hypothesize that force will correlate with established coagulation tests such as thrombelastography, demonstrate significant differences between healthy subjects and trauma patients, and identify critically ill trauma patients. METHODS: Blood samples were prospectively collected from level 1 trauma patients at initial presentation, assayed for force of and time to contraction and compared with thrombelastography. Blood from healthy subjects was assayed to establish a reference range. Results from trauma patients were compared with healthy controls and trauma patients that died. RESULTS: The study includes one hundred trauma patients with mean age 45 y, 74% were male, and median injury severity score of 14 ± 12. Patients that survived (n = 90) demonstrated significantly elevated platelet contraction force compared with healthy controls (n = 12) (6390 ± 2340 versus 4790 ± 470 µN, P = 0.043) and trauma patients that died (n = 10) (6390 ± 2340 versus 2860 ± 1830 µN, P = 0.0001). Elapsed time to start of platelet contraction was faster in trauma patients that survived compared with healthy controls (660 ± 467 versus 1130 ± 140 s, P = 0.0022) and those that died (660 ± 470 versus 1460 ± 1340 s, P < 0.0001). CONCLUSIONS: In contrast with all existing platelet function tests reported in the literature, which report platelet dysfunction in trauma patients, contractile force demonstrates hyperfunction in surviving trauma patients and dysfunction in nonsurvivors. Platelet contraction reflects platelet metabolic reserve and thus may be a potential biomarker for survival after trauma. Contractile force warrants further investigation to predict mortality in severely injured trauma patients.

Outcomes and prognostic factors of pediatric patients with a Glasgow Coma Score of 3 after blunt head trauma.

PURPOSE: This study aims to assess outcomes of pediatric patients with blunt traumatic brain injury (TBI) with a presenting Glasgow Coma Score (GCS) of 3. METHODS: After local institutional review board approval, we identified patients ages 0 to15 years with blunt TBI and a reported GCS of 3 between 2007 and 2017 from a pediatric level 1 trauma center prospective registry. Exclusion criteria were cardiac death on arrival and penetrating injury. We recorded clinical variables from patients with a non-pharmacologic GCS of 3 and pupillary exam documented by a neurosurgical attending or resident. The original Glasgow Outcome Scale (GOS) was used to compare with other studies. Importance of variables to survival was calculated. RESULTS: A total of 88 patients (mean age 6.9 years) were included with a mortality rate of 68%. Twelve percent had a poor long-term outcome (GOS 2 or 3) while 20% had a good long-term outcome (GOS 4 or 5). Median follow-up was 1.8 years. Initial group comparison revealed patients in group 1 (survivors) had less hypotension on arrival (14% SBP < 90 mmHg vs. 66%, p < 0.0001), higher temperatures on arrival (36.3 °C vs 34.9 °C, p = 0.0002), lower ISS (29.7 vs 39.5, p = 0.003), less serious injury to other major organs (34% vs 61%, p = 0.02), more epidural hematomas (24% vs 7%, p = 0.04), and less evidence of brain ischemia on CT (7% vs 39%, p = 0.002) or brainstem infarct, hemorrhage, or herniation (0% vs 27%, p = 0.002). Differences between the 2 groups in age, sex, race, MOI, AIS score, presence of midline shift > 5 mm, or time from injury to hospital arrival or time to surgery were not statistically significant. Classification tree analysis showed that the most important variable for survival was pupillary exam; mortality was 92% in presence of bilateral, fixed dilated pupils. The relative importance of initial temperature, MOI, and hypotension to survivability was 0.79, 0.75, and 0.47, respectively. CONCLUSION: Twenty percent of our pediatric non-pharmacologic GCS 3 cohort had a good functional outcome. Lack of bilaterally fixed and dilated pupils was the most important factor for survival. Temperature, MOI, and hypotension also correlated with survival. The data support selective aggressive management for these patients.

Pre-injury monocyte/macrophage depletion results in increased blood-brain barrier permeability after traumatic brain injury.

Traumatic brain injury (TBI) effects both the brain and the immune system. Circulating monocytes/macrophages (Mo /Ma ) after a TBI may play an important role in preserving the blood-brain barrier (BBB), reducing brain edema, and interacting with resident microglia. To elucidate the role of circulating Mo /Ma , we utilized a monocyte/macrophage depletion model in response to TBI in male rats. Clodronate liposomes (CL) were used to deplete circulating Mo /Ma . A controlled cortical impact (CCI) injury model was used to create a TBI. All animals received either CL or PBS liposomes (PL), 48 and 24 hr prior to the procedure, and were sacrificed 72 hr post-injury for analysis of BBB permeability, brain edema, whole blood (Mo /Ma and granulocytes), and/or microglial analysis. Animals undergoing Mo /Ma depletion with CL prior to CCI (CCI-CL) were found to have increased BBB permeability when compared to non-depleted CCI (CCI-PL) animals. At 72 hr following injury, Sham-CL maintained on average an 82% reduction in the whole blood monocytes when compared to Sham-PL (p < 0.001). Monocytes in the whole blood remained significantly lower in CCI-CL animals when compared to CCI-PL (p < 0.001). The number of granulocytes in the whole blood of CCI-CL animals was higher at 3 days when compared to CCI-PL (p < 0.022). Surprisingly, the depletion of Mo /Ma did not affect brain edema. However, the depletion of Mo /Ma did result in a significant decrease in microglia (CCI-CL vs. CCI-PL, p < 0.012). In conclusion, an intact Mo /Ma population is required to repair BBB integrity and microglial response following injury.

Inflammation-Stimulated Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Inflammation.

Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) have been proposed to be a key mechanistic link in the therapeutic efficacy of cells in response to cellular injuries through paracrine effects. We hypothesize that inflammatory stimulation of MSCs results in the release of EVs that have greater anti-inflammatory effects. The present study evaluates the immunomodulatory abilities of EVs derived from inflammation-stimulated and naive MSCs (MSCEv+ and MSCEv, respectively) isolated using a current Good Manufacturing Practice-compliant tangential flow filtration system. Detailed characterization of both EVs revealed differences in protein composition, cytokine profiles, and RNA content, despite similarities in size and expression of common surface markers. MSCEv+ further attenuated release of pro-inflammatory cytokines in vitro when compared to MSCEv, with a distinctly different pattern of EV-uptake by activated primary leukocyte subpopulations. The efficacy of EVs was partially attributed to COX2/PGE2 expression. The present study demonstrates that inflammatory stimulation of MSCs renders release of EVs that have enhanced anti-inflammatory properties partially due to COX2/PGE2 pathway alteration. Stem Cells 2018;36:79-90.