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.

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.

PET imaging of microglia using PBR28suv determines therapeutic efficacy of autologous bone marrow mononuclear cells therapy in traumatic brain injury.

Traumatic brain injury (TBI) results in activated microglia. Activated microglia can be measured in vivo by using positron emission topography (PET) ligand peripheral benzodiazepine receptor standardized uptake values (PBR28suv). Cell based therapies have utilized autologous bone marrow mononuclear cells (BMMNCs) to attenuate activated microglia after TBI. This study aims to utilize in vivo PBR28suv to assess the efficacy of BMMNCs therapy after TBI. Seventy-two hours after CCI injury, BMMNCs were harvested from the tibia and injected via tail-vein at 74 h after injury at a concentration of 2 million cells per kilogram of body weight. There were three groups of rats: Sham, CCI-alone and CCI-BMMNCs (AUTO). One hundred twenty days after injury, rodents were imaged with PBR28 and their cognitive behavior assessed utilizing the Morris Water Maze. Subsequent ex vivo analysis included brain volume and immunohistochemistry. BMMNCs therapy attenuated PBR28suv in comparison to CCI alone and it improved spatial learning as measured by the Morris Water Maze. Ex vivo analysis demonstrated preservation of brain volume, a decrease in amoeboid-shaped microglia in the dentate gyrus and an increase in the ratio of ramified to amoeboid microglia in the thalamus. PBR28suv is a viable option to measure efficacy of BMMNCs therapy after TBI.

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.

Autologous cellular therapy for cerebral palsy: a randomized, crossover trial.

We examined an autologous mononuclear-cell-therapy-based approach to treat cerebral palsy using autologous umbilical cord blood or bone-marrow-derived mononuclear cells. The primary objective was to determine if autologous cells are safe to administer in children with cerebral palsy. The secondary objectives were to determine if there was improvement in motor function of patients 12 months after infusion using the Gross Motor Function Measure and to evaluate impact of treatment on corticospinal tract microstructure as determined by radial diffusivity measurement. This Phase 1/2a trial was a randomized, blinded, placebo-controlled, crossover study in children aged 2-10 years of age with cerebral palsy enrolled between November 2013 and November 2016. Participants were randomized to 2:1 treatment:placebo. Treatment was either autologous bone-marrow-derived mononuclear cells or autologous umbilical cord blood. All participants who enrolled and completed their baseline visit planned to return for follow-up visits at 6 months, 12 months and 24 months after the baseline visit. At the 12-month post-treatment visit, participants who originally received the placebo received either bone-marrow-derived mononuclear cell or umbilical cord blood treatment. Twenty participants were included; 7 initially randomized to placebo, and 13 randomized to treatment. Five participants randomized to placebo received bone-marrow-derived mononuclear cells, and 2 received umbilical cord blood at the 12-month visit. None of the participants experienced adverse events related to the stem cell infusion. Cell infusion at the doses used in our study did not dramatically alter motor function. We observed concordant bilateral changes in radial diffusivity in 10 of 15 cases where each corticospinal tract could be reconstructed in each hemisphere. In 60% of these cases (6/10), concordant decreases in bilateral corticospinal tract radial diffusivity occurred post-treatment. In addition, 100% of unilateral corticospinal tract cases (3/3) exhibited decreased corticospinal tract radial diffusivity post-treatment. In our discordant cases (n = 5), directionality of changes in corticospinal tract radial diffusivity appeared to coincide with handedness. There was a significant improvement in corticospinal tract radial diffusivity that appears related to handedness. Connectivity strength increased in either or both pathways (corticio-striatal and thalamo-cortical) in each participant at 12 months post-treatment. These data suggest that both stem cell infusions are safe. There may be an improvement in myelination in some groups of patients that correlate with small improvements in the Gross Motor Function Measure scales. A larger autologous cord blood trial is impractical at current rates of blood banking. Either increased private banking or matched units would be required to perform a larger-scale trial.

Determining Sex-Based Differences in Inflammatory Response in an Experimental Traumatic Brain Injury Model.

Introduction: Traumatic brain injury is a leading cause of injury-related death and morbidity. Multiple clinical and pre-clinical studies have reported various results regarding sex-based differences in TBI. Our accepted rodent model of traumatic brain injury was used to identify sex-based differences in the pathological features of TBI. Methods: Male and female Sprague-Dawley rats were subjected to either controlled-cortical impact (CCI) or sham injury; brain tissue was harvested at different time intervals depending on the specific study. Blood-brain barrier (BBB) analysis was performed using infrared imaging to measure fluorescence dye extravasation. Microglia and splenocytes were characterized with traditional flow cytometry; microglia markers such as CD45, P2Y12, CD32, and CD163 were analyzed with t-distributed stochastic neighbor embedding (t-SNE). Flow cytometry was used to study tissue cytokine levels, and supplemented with ELISAs of TNF-⍺, IL-17, and IL-1ß of the ipsilateral hemisphere tissue. Results: CCI groups of both sexes recorded a higher BBB permeability at 72 hours post-injury than their respective sham groups. There was significant difference in the integrated density value of BBB permeability between the male CCI group and the female CCI group (female CCI mean = 3.08 x 108 ± 2.83 x 107, male CCI mean = 2.20 x 108 ± 4.05 x 106, p = 0.0210), but otherwise no differences were observed. Traditional flow cytometry did not distinguish any sex-based difference in regards to splenocyte cell population after CCI. t-SNE did not reveal any significant difference between the male and female injury groups in the activation of microglia. Cytokine analysis after injury by flow cytometry and ELISA was limited in differences at the time point of 6 hours post-injury. Conclusion: In our rodent model of traumatic brain injury, sex-based differences in pathology and neuroinflammation at specified time points are limited, and only noted in one specific analysis of BBB permeability.

A Case Report of Delayed Post-operative Angioedema Associated With Angiotensin-Converting Enzyme Inhibitor Use.

Angiotensin-converting enzyme inhibitors are known to precipitate angioedema. Drug-induced angioedema is rare in the perioperative setting. Even fewer cases described hours following a minor procedure. In this case report, we present a 45-year-old female who developed drug-induced angioedema hours following an obstetric procedure.

Neutrophil to Lymphocyte Ratio as a Predictor of Outcomes after Amputation.

BACKGROUND: The neutrophil-to-lymphocyte ratio (NLR) has become a prognostic marker for proinflammatory states. It is associated with outcomes in many clinical processes including critical limb ischemia. We sought to identify predictors of amputation failure and mortality, in addition to the role of NLR in patients undergoing above-knee amputations (AKAs) or below-knee amputations (BKAs). METHODS: All patients undergoing BKA or AKA between 2004 and 2014 at 3 institutions were identified and analyzed (n = 513). Patients were excluded if they did not have a complete blood count with differential within 7 days prior to their operations. Comparison groups were formed between patients requiring unplanned revision and those who did not, and additionally between survivors and nonsurvivors at 30 days postamputation. Patient demographics, intraoperative data, and postoperative courses were compared. A multinomial logistic regression model was created to further compare the groups. RESULTS: Four hundred and ten patients were included for analysis, of which 142 (35%) required unplanned revision. Nearly 5% of patients (19/410) died within 30 days of the initial amputation. On univariate analysis, those requiring revision were more likely to be current smokers compared to former smokers (P = 0.004 and P = 0.021, respectively), have a lower ankle-brachial index (ABI) (P = 0.019), and have undergone a BKA (P < 0.001). Patients with congestive heart failure (CHF) were less likely to require a revision after an amputation (P = 0.007). Postoperative NLR was higher in patients requiring revision (9.9 vs. 7.0, P < 0.001) and both preoperative and postoperative NLRs were higher in those with 30-day mortality (21.0 vs. 7.0, P < 0.001; 19.4 vs. 7.5, P < 0.001). A multinomial logistic regression model identified CHF (P = 0.004), ABI (P = 0.041), and elevated body mass index (BMI, P = 0.045) as predictors of revision, while coronary artery disease (CAD, P = 0.031), CHF (P = 0.029), and postoperative NLR (P < 0.001) were predictive of 30-day mortality. CONCLUSIONS: Postoperative elevated NLR, CAD, and CHF are predictors of 30-day mortality in patients undergoing major limb amputation, while CHF, elevated ABI, and high BMI are predictors of revision. This study suggests that NLR may have a role as a biomarker for poor outcomes in patients with underlying peripheral vascular disease and warrants further investigation.

Mynx Vascular Closure Device in Arterial Endovascular Procedures.

BACKGROUND: Vascular closure devices have been used to achieve hemostasis of percutaneous access sites following endovascular procedures, with reported decreased time for arterial control as well as decreased time to ambulation. We sought to determine rates and risk factors of postoperative bleeding complications and failures using these devices from a single institution experienced in the use of vascular closure devices. METHODS: All patients undergoing arterial endovascular procedures with percutaneous access between March 2010 and October 2015 at a single institution were identified and analyzed (n = 894). Patients undergoing endovascular aneurysm repair, open access, venous procedures, or upper extremity access were excluded. Comparison groups were formed between those using the Mynxgrip® (Mynx), Angio-Seal™, Perclose® vascular closure devices and manual pressure (MP). Patient demographics, intraoperative data, and postoperative complications were compared. RESULTS: A total of 615 (69%) patients received Mynx, 165 other vascular closure devices (VCD) ([14%] Perclose, 44 [4%] Angio-Seal), and 114 (13%) MP. MP patients were more likely to be diagnostic angiogram with smaller sheaths, while VCD patients were more likely to be interventions with larger sheaths. Univariate analysis identified age, atrial fibrillation, intervention (as opposed to diagnostic), and sheath size >5F associated with postoperative bleeding (P < 0.05), and in backward, logistic regression analysis, sheath size, age, and renal failure were independent predictors of the same. CONCLUSIONS: Use of vascular closure devices has a low rate of bleeding complication, device failure, and need for operative repair. Bleeding is associated with increased age, interventional procedure, and end-stage renal disease. Mynx, Perclose, and Angio-Seal have similar rates of complications. Use of these devices are a safe option for groin vessel closure.

Defining and Diagnosing Sepsis.

Sepsis is a heterogeneous clinical syndrome that encompasses infections of many different types and severity. Not surprisingly, it has confounded most attempts to apply a single definition, which has also limited the ability to develop a set of reliable diagnostic criteria. It is perhaps best defined as the different clinical syndromes produced by an immune response to infection that causes harm to the body beyond that of the local effects of the infection.

Congestive Heart Failure.

Patients with acute decompensated heart failure are usually critically ill and require immediate treatment. However, most are not volume overloaded. Emergency department (ED) management is based on rapid initiation of noninvasive positive-pressure ventilation and aggressive titration of nitrates. Afterload reduction with an angiotensin-converting enzyme inhibitor can be considered. A diuretic should not be administered before optimal preload and afterload reduction has been achieved. Short-term inotropic therapy can be considered in select patients with cardiogenic shock and acute decompensated heart failure (ADHF) who fail to respond to standard therapy.

High initial tidal volumes in emergency department patients at risk for acute respiratory distress syndrome.

PURPOSE: Emergency department (ED) patients are at high risk for the acute respiratory distress syndrome (ARDS). Settings only 1 mL/kg above recommended tidal volumes confers harm for these patients. The purpose of this study was to determine whether ED physicians routinely initiate mechanical ventilation with low tidal volumes in patients at risk for ARDS. MATERIALS AND METHODS: We retrospectively reviewed the charts of all adult patients who were intubated in an urban, academic ED. The charts were analyzed to identify patients in whom ARDS developed within 48 hours after ED admission. Patients were eligible for inclusion if they had bilateral infiltrates on imaging, had a Pao2/Fio2 ratio less than 300 mm Hg and did not have heart failure contributing to their presentation. The tidal volumes set in the ED were then compared with the recommended tidal volume of 6 mL/kg of predicted body weight. RESULTS: The initial tidal volumes set in the ED were higher than recommended by an average of 80 mL (95% confidence interval, 60-110, P < .0001) or 1.5 mL/kg (95% confidence interval, 1.0-1.9). Only 5 of the 34 patients received the recommended tidal volume ventilation setting. CONCLUSIONS: In an academic, tertiary hospital, newly intubated ED patients in whom ARDS developed within 48 hours after intubation were ventilated with tidal volumes that exceeded recommendations by an average of 1.5 mL/kg.

Assessing volume status.

Shock is a physiologic state associated with high morbidity and mortality rates. The clinician has several tools available to evaluate volume status. Each modality has its benefits and limitations but, to date, no one test can indicate with 100% accuracy which patients will be truly volume responsive. Although the search for the Holy Grail of a perfect intravascular monitor continues, we must remember the importance of early, aggressive, and goal-directed interventions for patients in shock. Finally, there is no substitute for the most important intervention-the frequent presence of the physician at the patient's bedside.