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.

Time dependent analysis of rat microglial surface markers in traumatic brain injury reveals dynamics of distinct cell subpopulations.

Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to the activation of microglia. Accurate identification of microglial populations is key to understanding therapeutic approaches that modify microglial responses to TBI and improve long-term outcome measures. Notably, previous studies often utilized an outdated convention to describe microglial phenotypes. We conducted a temporal analysis of the response to controlled cortical impact (CCI) in rat microglia between ipsilateral and contralateral hemispheres across seven time points, identified microglia through expression of activation markers including CD45, CD11b/c, and p2y12 receptor and evaluated their activation state using additional markers of CD32, CD86, RT1B, CD200R, and CD163. We identified unique sub-populations of microglial cells that express individual or combination of activation markers across time points. We further portrayed how the size of these sub-populations changes through time, corresponding to stages in TBI response. We described longitudinal changes in microglial population after CCI in two different locations using activation markers, showing clear separation into cellular sub-populations that feature different temporal patterns of markers after injury. These changes may aid in understanding the symptomatic progression following TBI and help define microglial subpopulations beyond the outdated M1/M2 paradigm.

Human-derived Treg and MSC combination therapy may augment immunosuppressive potency in vitro, but did not improve blood brain barrier integrity in an experimental rat traumatic brain injury model.

Traumatic brain injury (TBI) causes both physical disruption of the blood brain barrier (BBB) and altered immune responses that can lead to significant secondary brain injury and chronic inflammation within the central nervous system (CNS). Cell therapies, including mesenchymal stromal cells (MSC), have been shown to restore BBB integrity and augment endogenous splenic regulatory T cells (Treg), a subset of CD4+ T cells that function to regulate immune responses and prevent autoimmunity. We have recently shown that infusion of human cord blood-derived Treg decreased neuroinflammation after TBI in vivo and in vitro. However, while both cells have demonstrated anti-inflammatory and regenerative potential, they likely utilize differing, although potentially overlapping, mechanisms. Furthermore, studies investigating these two cell types together, as a combination therapy, are lacking. In this study, we compared the ability of Treg+MSC combination therapy, as well as MSC and Treg monotherapies, to improve BBB permeability in vivo and suppress inflammation in vitro. While Treg+MSC combination did not significantly augment potency in vivo, our in vitro data demonstrates that combination therapy may augment therapeutic potency and immunosuppressive potential compared to Treg or MSC monotherapy.

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.

High-resolution and differential analysis of rat microglial markers in traumatic brain injury: conventional flow cytometric and bioinformatics analysis.

Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to microglial activation. Transforming from surveying to primed phenotypes, microglia undergo considerable molecular changes. However, specific microglial profiles in rat remain elusive due to tedious methodology and limited availability of reagents. Here, we present a flow cytometry-based analysis of rat microglia 24 h after TBI using the controlled cortical impact model, validated with a bioinformatics approach. Isolated microglia are analyzed for morphological changes and their expression of activation markers using flow cytometry, traditional gating-based analysis methods and support the data by employing bioinformatics statistical tools. We use CD45, CD11b/c, and p2y12 receptor to identify microglia and evaluate their activation state using CD32, CD86, RT1B, CD200R, and CD163. The results from logic-gated flow cytometry analysis was validated with bioinformatics-based analysis and machine learning algorithms to detect quantitative changes in morphology and marker expression in microglia due to activation following TBI.

Human cord blood-derived regulatory T-cell therapy modulates the central and peripheral immune response after traumatic brain injury.

Traumatic brain injury (TBI) causes a profound inflammatory response within the central nervous system and peripheral immune system, which contributes to secondary brain injury and further morbidity and mortality. Preclinical investigations have demonstrated that treatments that downregulate microglia activation and polarize them toward a reparative/anti-inflammatory phenotype have improved outcomes in preclinical models. However, no therapy to date has translated into proven benefits in human patients. Regulatory T cells (Treg) have been shown to downregulate pathologic immune responses of the innate and adaptive immune system across a variety of pathologies. Furthermore, cellular therapy has been shown to augment host Treg responses in preclinical models; yet, studies investigating the use of Treg as a therapeutic for TBI are lacking. In a rodent TBI model, we demonstrate that human umbilical cord blood Treg modulate the central and peripheral immune response after injury in vitro and in vivo.

Resuscitation Strategies for Traumatic Brain Injury.

PURPOSE OF REVIEW: Traumatic brain injury (TBI) is a leading cause of morbidity and mortality; however, little definitive evidence exists about most clinical management strategies. Here, we highlight important differences between two major guidelines, the 2016 Brain Trauma Foundation guidelines and the Lund Concept, along with recent pre-clinical and clinical data. RECENT FINDINGS: While intracranial pressure (ICP) monitoring has been questioned, the majority of literature demonstrates benefit in severe TBI. The optimal cerebral perfusion pressure (CPP) and ICP are yet unknown, but likely as important is the concept of ICP burden. The evidence for anti-hypertensive therapy is strengthening. Decompressive craniectomy improves mortality, but at the cost of increased morbidity. Plasma-based resuscitation has demonstrated benefit in multiple pre-clinical TBI studies. SUMMARY: The management of hemodynamics and intravascular volume are crucial in TBI. Based on recent evidence, ICP monitoring, anti-hypertensive therapy, minimal use of vasopressors/inotropes, and plasma resuscitation may improve outcomes.

Therapeutic time window of multipotent adult progenitor therapy after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability. TBI results in a prolonged secondary central neuro-inflammatory response. Previously, we have demonstrated that multiple doses (2 and 24 h after TBI) of multipotent adult progenitor cells (MAPC) delivered intravenously preserve the blood-brain barrier (BBB), improve spatial learning, and decrease activated microglia/macrophages in the dentate gyrus of the hippocampus. In order to determine if there is an optimum treatment window to preserve the BBB, improve cognitive behavior, and attenuate the activated microglia/macrophages, we administered MAPC at various clinically relevant intervals. METHODS: We administered two injections intravenously of MAPC treatment at hours 2 and 24 (2/24), 6 and 24 (6/24), 12 and 36 (12/36), or 36 and 72 (36/72) post cortical contusion injury (CCI) at a concentration of 10 million/kg. For BBB experiments, animals that received MAPC at 2/24, 6/24, and 12/36 were euthanized 72 h post injury. The 36/72 treated group was harvested at 96 h post injury. RESULTS: Administration of MAPC resulted in a significant decrease in BBB permeability when administered at 2/24 h after TBI only. For behavior experiments, animals were harvested post behavior paradigm. There was a significant improvement in spatial learning (120 days post injury) when compared to cortical contusion injury (CCI) in groups when MAPC was administered at or before 24 h. In addition, there was a significant decrease in activated microglia/macrophages in the dentate gyrus of hippocampus of the treated group (2/24) only when compared to CCI. CONCLUSIONS: Intravenous injections of MAPC at or before 24 h after CCI resulted in improvement of the BBB, improved cognitive behavior, and attenuated activated microglia/macrophages in the dentate gyrus.

Prevalence and Impact of Admission Acute Traumatic Coagulopathy on Treatment Intensity, Resource Use, and Mortality: An Evaluation of 956 Severely Injured Children and Adolescents.

BACKGROUND: Acute coagulopathy of trauma in children is of potential importance to clinical outcomes, but knowledge is limited and has only been investigated using conventional coagulation testing. The purpose of this study was to assess the prevalence and impact of arrival coagulopathy, determined by viscoelastic hemostatic testing, in severely injured children. STUDY DESIGN: Pediatric patients (younger than 17 years of age) who were admitted January 2010 to May 2016 and met highest-level trauma activation were included. Patients were divided into 2 groups (coagulopathy and controls) based on arrival rapid thrombelastography values. Coagulopathy was defined as the presence of any of the following on rapid thrombelastography: activated clotting time ≥128 seconds, α-angle ≤65 degrees, maximum amplitude ≤55 mm, and lysis at 30 minutes from 20-mm amplitude ≥3%. Logistic regression was used to adjust for age, sex, blood pressure, mechanism, and injury severity. RESULTS: Nine hundred and fifty-six patients met inclusion; 507 (57%) were coagulopathic and 449 (43%) were not (noncoagulopathic and control cohort). Coagulopathic patients were younger (median 14 vs 15 years) and more likely to be male (68% vs 60%) and Hispanic (38% vs 31%) (all p < 0.05). Coagulopathic patients received more RBC and plasma transfusions and had fewer ICU and ventilator-free days and higher mortality (12% vs 3%; all p < 0.05). Of these 956, 197 (21%) sustained severe brain injury-123 (62%) were coagulopathic and 74 (38%) were noncoagulopathic. The mortality difference was even greater for coagulopathic head injuries (31% vs 10%; p = 0.002). Adjusting for confounders, admission coagulopathy was an independent predictor of death, with an odds ratio of 3.67 (95% CI 1.768 to 7.632; p < 0.001). CONCLUSIONS: Almost 60% of severely injured children and adolescents arrive with evidence of acute traumatic coagulopathy. The presence of admission coagulopathy is associated with high mortality in children, especially among those with head injuries.

Do microglia play a role in sex differences in TBI?

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality for both males and females and is, thus, a major focus of current study. Although the overall death rate of TBI for males is roughly three times higher than that for females, males have been disproportionately represented in clinical and preclinical studies. Gender differences are known to exist in many neurologic disorders, such as multiple sclerosis and stroke, and differences appear to exist in TBI. Furthermore, it is known that microglia have sexually dimorphic roles in CNS development and other neurologic conditions; however, most animal studies of microglia and TBI have focused on male subjects. Microglia are a current target of many preclinical and clinical therapeutic trials for TBI. Understanding the relationship among sex, sex hormones, and microglia is critical to truly understanding the pathophysiology of TBI. However, the evidence for sex differences in TBI centers mainly on sex hormones, and evidenced-based conclusions are often contradictory. In an attempt to review the current literature, it is apparent that sex differences likely exist, but the contradictory nature and magnitude of such differences in the existing literature does not allow definite conclusions to be drawn, except that more investigation of this issue is necessary. © 2016 Wiley Periodicals, Inc.