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Traumatic brain injury (TBI) patients frequently develop cardiopulmonary system complications such as acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). However, the mechanism by which TBI causes ALI/ARDS is not fully understood. Here, we used a severe TBI model to examine the effects of a low-molecular-weight heparin, enoxaparin, on inflammasome activation and lung injury damage. We investigated whether enoxaparin inhibits ALI and inflammasome signaling protein expression in the brain and lungs after TBI in mice. C57/BL6 mice were subjected to severe TBI and were treated with vehicle or 1 mg/kg of enoxaparin 30 min after injury. Lung and brain tissue were collected 24 h post-TBI and were analyzed by immunoblotting for expression of the inflammasome proteins, caspase-1 and interleukin (IL)-1β. In addition, lung tissue was collected for histological analysis to determine ALI scoring and neutrophil and macrophage infiltration post-injury. Our data show that severe TBI induces increased expression of inflammasome proteins caspase-1 and IL-1β in the brain and lungs of mice after injury. Treatment with enoxaparin attenuated inflammasome expression in the brain and lungs 24 h after injury. Enoxaparin significantly decreased ALI score as well as neutrophil and macrophage infiltration in lungs at 24 h after injury. This study demonstrates that enoxaparin attenuates ALI and inhibits inflammasome expression in the brain and lungs after TBI. These findings support the hypothesis that inhibition of the neural-respiratory inflammasome axis that is activated after TBI may have therapeutic potential.

Significance: Low frequency whole body vibration (LFV) at 40 Hz, a low impact form of exercise, for a month following mild transient middle-cerebral artery occlusion (tMCAO) reduces infarct volume and improves motor function in reproductively senescent, middle-aged female rats (1). In humans, LFV was shown to increase circulating levels of irisin, a skeletal muscle-derived hormone. Irisin has also been shown to play a crucial role in preserving mitochondrial function, preventing oxidative stress, and elevating expression of BDNF, among other neuroprotective measures. The current study aims to investigate the efficacy of LFV in ameliorating post-tMCAO cognitive deficits and to determine the putative role of irisin in conferring the benefits of LFV in middle-aged rats. Methods: Middle-aged rats of both sexes (5-8) were randomly assigned to tMCAO (90 min), or sham surgery followed by exposure to either LFV (twice a day for 15 min each for 5 days a week over a month) or no LFV treatment groups. Following the last LFV treatment, rats were tested for hippocampus-dependent learning and memory using a water maze followed harvesting brain and blood samples for histopathological and inflammatory marker analyses, respectively. In a parallel experiment in the absence of LFV, middle-aged female rats were randomly assigned to either saline or irisin treatment following tMCAO. Recombinant irisin was purchased from PeproTech (Rocky Hill, NJ). Rats were treated with irisin (0.2 μg/g BW; IP) or saline for a month followed by their brains were assessed by histopathology. Results: Post-tMCAO LFV significantly lessens cognitive deficits in rats of both sexes. It also significantly decreased circulating pro-inflammatory cytokines and increased serum levels of native irisin. Quantification of infarct volume irisin-treated rats demonstrated that, compared to saline, infarct volume was significantly reduced. Saline treatment resulted in 234 ± 30 mm 3 , while irisin treatment yielded 128 ± 34 mm 3 (p Conclusion: Irisin, either as elicited by LFV or administered exogenously without LFV, regulates brain metabolism and inflammation and its beneficial effects can be stimulated by LFV.Reference: 1. Raval, A.P., et al., Int J Mol Sci, 2018. 19 (9).

Ischemic stroke kills more women compared with men thus emphasizing a significant sexual dimorphism in ischemic pathophysiological outcomes. However, the mechanisms behind this sexual dimorphism are yet to be fully understood. It is well established that cerebral ischemia activates a variety of inflammatory cascades and that microglia are the primary immune cells of the brain. After ischemic injury, microglia are activated and play a crucial role in progression and resolution of the neuroinflammatory response. In recent years, research has focused on the role that microglia play in this sexual dimorphism that exists in the response to central nervous system (CNS) injury. Evidence suggests that the molecular mechanisms leading to microglial activation and polarization of phenotypes may be influenced by sex, therefore causing a difference in the pro/anti‐inflammatory responses after CNS injury. Here, we review advances highlighting that sex differences in microglia are an important factor in the inflammatory responses that are seen after ischemic injury. We discuss the main differences between microglia in the healthy and diseased developing, adult, and aging brain. We also focus on the dimorphism that exists between males and females in microglial‐induced inflammation and energy metabolism after CNS injury. Finally, we describe how all of the current research and literature regarding sex differences in microglia contribute to the differences in poststroke responses between males and females.

Hyperoxia-induced lung injury plays a key role in the development of bronchopulmonary dysplasia (BPD), characterized by inflammatory injury and impaired lung development in preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, currently it is uncertain how lung injury contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that regulate intercellular and inter-organ communications. Gasdermin D (GSDMD) has emerged as a key executor of inflammasome-mediated cell death and inflammation. In this study, we utilized a neonatal rat model of BPD to assess if hyperoxia stimulates lung release of circulating EVs and if these EVs induce lung and brain injury. We found that hyperoxia-exposed rats had elevated numbers of plasma-derived EVs compared to rats maintained in room air. These EVs also had increased cargos of surfactant protein C, a marker of type II alveolar epithelial cells (AEC), and the active (p30) form of GSDMD. When these EVs were adoptively transferred into normal newborn rats via intravenous injection, they were taken up both by lung and brain tissues. Moreover, EVs from hyperoxic animals induced not only the pathological hallmarks of BPD, but also brain inflammatory injury in recipient rats, as well as inducing cell death in cultured pulmonary vascular endothelial cells and neural stem cells (NSC). Similarly, hyperoxia-exposed cultured AEC-like cells released EVs that also contained increased GSDMD-p30 and these EVs induced pyroptotic cell death in NSC. Overall, these data indicate that hyperoxia-activated circulating EVs mediate a lung to brain crosstalk resulting in brain injury and suggest a mechanism that links lung injury and neurodevelopmental impairment in BPD infants.

Approximately 20-25% of traumatic brain injury (TBI) subjects develop acute lung injury (ALI), but the pathomechanisms of TBI-induced ALI remain poorly defined. Our previous work has shown that the inflammasome plays a critical role in TBI-induced secondary pathophysiology and that inflammasome proteins are released in extracellular vesicles (EV) after TBI. Here we investigated whether EV-mediated inflammasome signaling contributed to the etiology of TBI-induced ALI. C57/BL6 male mice were subjected to controlled cortical impact (CCI), and the brains and lungs were examined for inflammasome activation and ALI at 4 and 24 h after TBI. We show that TBI releases EV containing inflammasome proteins into serum that target the lung to cause ALI, supporting activation of a neural-respiratory-inflammasome axis. Administration of a low-molecular-weight heparin (enoxaparin, a blocker of EV uptake) or treatment with a monoclonal antibody against apoptosis speck-like staining protein containing a caspase recruitment domain (anti-ASC) after adoptive transfer of EV isolated from TBI-injured mice significantly inhibited inflammasome activation in the lungs of recipient mice resulting in improved ALI scores.This axis constitutes an important arm of the innate inflammatory response in lung pathology after TBI and targeting this axis represents a novel therapeutic treatment for TBI-induced ALI.

Hyperoxia plays a key role in the development of bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants. Infants with BPD often have brain injury that leads to long-term neurodevelopmental impairment, but the underlying mechanisms that control BPD-induced neurodevelopmental impairment remain unclear. Our previous studies have shown that hyperoxia-induced BPD in rodents is associated with lung inflammasome activation. Here, we tested the hypothesis that hyperoxia-induced lung and brain injury is mediated by inflammasome activation, and that inhibition of caspase-1, a key component of the inflammasome, attenuates hyperoxia-induced lung and brain injury in neonatal mice. C57/BL6 mouse pups were randomized to receive daily intraperitoneal injections of Ac-YVAD-CMK, an irreversible caspase-1 inhibitor, or placebo during exposure to room air or hyperoxia (85% O2) for 10 days. We found that hyperoxia activated the NLRP1 inflammasome, increased production of mature IL-1β, and upregulated expression of p30 gasdermin-D (GSDMD), the active form of GSDMD that is responsible for the programmed cell death mechanism of pyroptosis in both lung and brain tissue. Importantly, we show that inhibition of caspase-1 decreased IL-1β activation and p30 GSDMD expression, and improved alveolar and vascular development in hyperoxia-exposed lungs. Moreover, caspase-1 inhibition also promoted cell proliferation in the subgranular zone and subventricular zone of hyperoxia-exposed brains, resulting in lessened atrophy of these zones. Thus, the inflammasome plays a critical role in hyperoxia-induced neonatal lung and brain injury, and targeting this pathway may be beneficial for the prevention of lung and brain injury in preterm infants.

Approximately 30% of traumatic brain injured patients suffer from acute lung injury or acute respiratory distress syndrome. Our previous work revealed that extracellular vesicle (EV)-mediated inflammasome signaling plays a crucial role in the pathophysiology of traumatic brain injury (TBI)-induced lung injury. Here, serum-derived EVs from severe TBI patients were analyzed for particle size, concentration, origin, and levels of the inflammasome component, an apoptosis-associated speck-like protein containing a caspase-recruiting domain (ASC). Serum ASC levels were analyzed from EV obtained from patients that presented lung injury after TBI and compared them to EV obtained from patients that did not show any signs of lung injury. EVs were co-cultured with lung human microvascular endothelial cells (HMVEC-L) to evaluate inflammasome activation and endothelial cell pyroptosis. TBI patients had a significant increase in the number of serum-derived EVs and levels of ASC. Severe TBI patients with lung injury had a significantly higher level of ASC in serum and serum-derived EVs compared to individuals without lung injury. Only EVs isolated from head trauma patients with gunshot wounds were of neural origin. Delivery of serum-derived EVs to HMVEC-L activated the inflammasome and resulted in endothelial cell pyroptosis. Thus, serum-derived EVs and inflammasome proteins play a critical role in the pathogenesis of TBI-induced lung injury, supporting activation of an EV-mediated neural-respiratory inflammasome axis in TBI-induced lung injury.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality. Approximately 20-25 percent of TBI subjects develop Acute Lung Injury (ALI), but the pathomechanisms of TBI-induced ALI remain poorly defined. Currently, mechanical ventilation is the only therapeutic intervention for TBI-induced lung injury. Our recent studies have shown that the inflammasome plays an important role in the systemic inflammatory response leading to lung injury-post TBI. Here, we outline the role of the extracellular vesicle (EV)-mediated inflammasome signaling in the etiology of TBI-induced ALI. Furthermore, we evaluate the efficacy of a low molecular weight heparin (Enoxaparin, a blocker of EV uptake) and a monoclonal antibody against apoptosis speck-like staining protein containing a caspase recruitment domain (anti-ASC) as therapeutics for TBI-induced lung injury. We demonstate that activation of an EV-mediated Neural-Respiratory Inflammasome Axis plays an essential role in TBI-induced lung injury and disruption of this axis has therapeutic potential as a treatment strategy.

Background The inflammasome plays an important role in the inflammatory innate immune response after central nervous system (CNS) injury. Inhibition of the inflammasome after traumatic brain injury (TBI) results in improved outcomes by lowering the levels of caspase-1 and interleukin (IL)-1b. We have previously shown that inflammasome proteins are elevated in the cerebrospinal fluid (CSF) of patients with TBI and that higher levels of these proteins were consistent with poorer outcomes after TBI when compared to patients that presented these inflammasome proteins at lower levels. Methods and findings Here we extend our work by analyzing serum from 21 TBI patients and CSF from 18 TBI patients compared to 120 serum samples and 30 CSF samples from no-TBI donor controls for the expression of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), interleukin(IL)-1b and IL-18. Analysis was carried out using the Ella Simple Plex system (Protein Simple) to determine the sensitivity and specificity of inflammasome proteins as biomarkers of TBI. Receiver operator characteristic (ROC) curves, confidence intervals and likelihood ratios for each biomarker was determined. ROC curves, confidence intervals, sensitivity and specificity for each biomarker examined revealed that caspase-1 (0.93 area under the curve (AUC)) and ASC (0.90 AUC) in serum and ASC (1.0 AUC) and IL-18 (0.84 AUC) in CSF are promising biomarkers of TBI pathology. Importantly, higher protein levels (above 547.6 pg/ml) of ASC (0.91 AUC) were consistent with poorer outcomes after TBI as determined by the Glasgow Outcome Scale-Extended (GOSE). Conclusion These findings indicate that inflammasome proteins are excellent diagnostic and predictive biomarkers of TBI., This work was supported by a STTR grant (4R42NS086274-012) from the NINDS/NIH to RWK: https://www.nih.gov/; and by a public grant from Fondo de Investigacion Sanitaria (FIS PI16/00737) to JPB: https://portalfis.isciii.es/es/Paginas/inicio.aspx.No funding bodies had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.; This work was supported by a STTR grant (4R42NS086274-012) from the NINDS/NIH to RWK and WDD and by a public grant from Fondo de Investigacion Sanitaria (FIS PI16/00737) to JPB.

Background: Inflammatory injury, particularly the production of active interleukin (IL)-1β plays a major role in the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. The release of active IL-1β is controlled by posttranscriptional modifications of its proform (pro-IL-1β) through the inflammasome. Rac1 is a member of the Rho family of GTPases that regulate the inflammatory process. Objective: This study tested the hypothesis that Rac1 signaling increases inflammasome activation that results in damaging inflammation, and that the inhibition of Rac1 signaling prevents lung injury, by inhibiting inflammasome activation in a newborn rat model of BPD induced by hyperoxia. Methods: Newborn rat pups were exposed to room air or hyperoxia (85% O2) and received daily intraperitoneal injections of placebo (normal saline) or NSC23766, a specific Rac1 inhibitor, for 10 days. The effects on lung inflammation, alveolarization, vascular development, vascular remodeling, right ventricular systolic pressure, and right ventricular hypertrophy (RVH) were then assessed. Results: Hyperoxia exposure upregulated Rac1 and increased the production of active IL-1β, which was accompanied by increasing expression of the inflammasome. In addition, hyperoxia induced the pathological hallmarks of BPD. However, treatment with NSC23766 significantly decreased inflammasome activation and macrophage infiltration, improved alveolar and vascular development, and reduced pulmonary vascular remodeling and RVH. Conclusion: These results indicate that Rac1 signaling regulates the expression of the inflammasome and plays a pivotal role in the pathogenesis of hyperoxia-induced neonatal lung injury. Therefore, targeting Rac1 signaling may provide a novel strategy to prevent and treat BPD in preterm infants.