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Traumatic brain injury (TBI) can induce systemic coagulopathy and inflammation, thereby increasing the risk of mortality and disability. However, the mechanism causing systemic coagulopathy and inflammation following TBI remains unclear. In prior research, we discovered that brain-derived extracellular vesicles (BDEVs), originating from the injured brain, can activate the coagulation cascade and inflammatory cells. In this study, we primarily investigated how BDEVs affect systemic coagulopathy and inflammation in peripheral circulation. The results of cytokines and coagulation function indicated that BDEVs can lead to systemic coagulopathy and inflammation by influencing inflammatory factors and chemokines within 24 h. Furthermore, according to flow cytometry and blood cell counter results, we found that BDEVs induced changes in the blood count such as a reduced number of platelets and leukocytes and an increased percentage of neutrophils, macrophages, activated platelets, circulating platelet-EVs, and leukocyte-derived EVs. We also discovered that eliminating circulating BDEVs with lactadherin helped improve coagulopathy and inflammation, relieved blood cell dysfunction, and decreased the circulating platelet-EVs and leukocyte-derived EVs. Our research provides a novel viewpoint and potential mechanism of TBI-associated secondary damage., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Neuroinflammation is one of the most important pathogeneses in secondary brain injury after traumatic brain injury (TBI). Neutrophil extracellular traps (NETs) forming neutrophils were found throughout the brain tissue of TBI patients and elevated plasma NET biomarkers correlated with worse outcomes. However, the biological function and underlying mechanisms of NETs in TBI-induced neural damage are not yet fully understood. Here, we used Cl-amidine, a selective inhibitor of NETs to investigate the role of NETs in neural damage after TBI., Methods: Controlled cortical impact model was performed to establish TBI. Cl-amidine, 2'3'-cGAMP (an activator of stimulating Interferon genes (STING)), C-176 (a selective STING inhibitor), and Kira6 [a selectively phosphorylated inositol-requiring enzyme-1 alpha [IRE1α] inhibitor] were administrated to explore the mechanism by which NETs promote neuroinflammation and neuronal apoptosis after TBI. Peptidyl arginine deiminase 4 (PAD4), an essential enzyme for neutrophil extracellular trap formation, is overexpressed with adenoviruses in the cortex of mice 1 day before TBI. The short-term neurobehavior tests, magnetic resonance imaging (MRI), laser speckle contrast imaging (LSCI), Evans blue extravasation assay, Fluoro-Jade C (FJC), TUNEL, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative-PCR were performed in this study., Results: Neutrophils form NETs presenting in the circulation and brain at 3 days after TBI. NETs inhibitor Cl-amidine treatment improved short-term neurological functions, reduced cerebral lesion volume, reduced brain edema, and restored cerebral blood flow (CBF) after TBI. In addition, Cl-amidine exerted neuroprotective effects by attenuating BBB disruption, inhibiting immune cell infiltration, and alleviating neuronal death after TBI. Moreover, Cl-amidine treatment inhibited microglia/macrophage pro-inflammatory polarization and promoted anti-inflammatory polarization at 3 days after TBI. Mechanistically, STING ligand 2'3'-cGAMP abolished the neuroprotection of Cl-amidine via IRE1α/ASK1/JNK signaling pathway after TBI. Importantly, overexpression of PAD4 promotes neuroinflammation and neuronal death via the IRE1α/ASK1/JNK signaling pathway after TBI. However, STING inhibitor C-176 or IRE1α inhibitor Kira6 effectively abolished the neurodestructive effects of PAD4 overexpression after TBI., Conclusion: Altogether, we are the first to demonstrate that NETs inhibition with Cl-amidine ameliorated neuroinflammation, neuronal apoptosis, and neurological deficits via STING-dependent IRE1α/ASK1/JNK signaling pathway after TBI. Thus, Cl-amidine treatment may provide a promising therapeutic approach for the early management of TBI., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Traumatic brain injury (TBI) is an important reason of neurological damage and has high morbidity and mortality rates. The secondary damage caused by TBI leads to a poor clinical prognosis. According to the literature, TBI leads to ferrous iron aggregation at the site of trauma and may be a key factor in secondary injury. Deferoxamine (DFO), which is an iron chelator, has been shown to inhibit neuron degeneration; however, the role of DFO in TBI is unclear. The purpose of this study was to explore whether DFO can ameliorate TBI by inhibiting ferroptosis and neuroinflammation. Here, our findings suggest that DFO can reduce the accumulation of iron, lipid peroxides, and reactive oxygen species (ROS) and modulate the expression of ferroptosis-related indicators. Moreover, DFO may reduce NLRP3 activation via the ROS/NF-κB pathway, modulate microglial polarization, reduce neutrophil and macrophage infiltration, and inhibit the release of inflammatory factors after TBI. Additionally, DFO may reduce the activation of neurotoxic responsive astrocytes. Finally, we demonstrated that DFO can protect motor memory function, reduce edema and improve peripheral blood perfusion at the site of trauma in mice with TBI, as shown by behavioral experiments such as the Morris water maze test, cortical blood perfusion assessment and animal MRI. In conclusion, DFO ameliorates TBI by reducing iron accumulation to alleviate ferroptosis and neuroinflammation, and these findings provide a new therapeutic perspective for TBI., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Endoplasmic reticulum (ER) stress and ER stress-mediated apoptosis play an important role during secondary brain damage after traumatic brain injury (TBI). Increased neutrophil extracellular traps (NETs) formation has been demonstrated to be associated with neurological damage after TBI. However, the correlation between ER stress and NETs remains unclear, and the specific function of NETs in neurons has not been defined. In this study, we found that the levels of NETs circulating biomarkers were remarkably elevated in the plasma of TBI patients. We then inhibited NETs formation by peptidylarginine deiminase 4 (PAD4, a critical enzyme for NETs formation) deficiency and discovered that ER stress activation and ER stress-mediated neuronal apoptosis were reduced. The degradation of NETs via DNase I showed similar outcomes. Furthermore, overexpression of PAD4 aggravated neuronal ER stress and ER stress-associated apoptosis, while TLR9 antagonist administration abrogated the damage caused by NETs. In addition to in vivo experiments, in vitro experiments revealed that treatment with a TLR9 antagonist alleviated NETs-induced ER stress and apoptosis in HT22 cells. Collectively, our results indicated that ER stress as well as the accompanying neuronal apoptosis can be ameliorated by disruption of NETs and that suppression of the TLR9-ER stress signaling pathway may contribute to positive outcomes after TBI., (© 2023. The Author(s).)

Introduction: Increased neutrophil extracellular trap (NET) formation has been reported to be associated with cerebrovascular dysfunction and neurological deficits in traumatic brain injury (TBI). However, the biological function and underlying mechanisms of NETs in TBI-induced neuronal cell death are not yet fully understood., Methods: First, brain tissue and peripheral blood samples of TBI patients were collected, and NETs infiltration in TBI patients was detected by immunofluorescence staining and Western blot. Then, a controlled cortical impact device was used to model brain trauma in mice, and Anti-Ly6G, DNase, and CL-amidine were given to reduce the formation of neutrophilic or NETs in TBI mice to evaluate neuronal death and neurological function. Finally, the pathway changes of neuronal pyroptosis induced by NETs after TBI were investigated by administration of peptidylarginine deiminase 4 (a key enzyme of NET formation) adenovirus and inositol-requiring enzyme-1 alpha (IRE1α) inhibitors in TBI mice., Results: We detected that both peripheral circulating biomarkers of NETs and local NETs infiltration in the brain tissue were significantly increased and had positive correlations with worse intracranial pressure (ICP) and neurological dysfunction in TBI patients. Furthermore, the depletion of neutrophils effectively reduced the formation of NET in mice subjected to TBI. we found that degradation of NETs or inhibition of NET formation significantly inhibited nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 1 (NLRP1) inflammasome-mediated neuronal pyroptosis after TBI, whereas these inhibitory effects were abolished by cyclic GMP-AMP (cGAMP), an activator of stimulating Interferon genes (STING). Moreover, overexpression of PAD4 in the cortex by adenoviruses could aggravate NLRP1-mediated neuronal pyroptosis and neurological deficits after TBI, whereas these pro-pyroptotic effects were rescued in mice also receiving STING antagonists. Finally, IRE1α activation was significantly upregulated after TBI, and NET formation or STING activation was found to promote this process. Notably, IRE1α inhibitor administration significantly abrogated NETs-induced NLRP1 inflammasome-mediated neuronal pyroptosis in TBI mice., Discussion: Our findings indicated that NETs could contribute to TBI-induced neurological deficits and neuronal death by promoting NLRP1-mediated neuronal pyroptosis. Suppression of the STING/ IRE1α signaling pathway can ameliorate NETs-induced neuronal pyroptotic death after TBI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cao, Shi, Liu, Zuo, Jia, Min, Liu, Chen, Zhou, Li, Yang, Liu, Deng, Chen, Chen, Zhang and Zhang.)

Background and Purpose: Neuroinflammation has been shown to play a critical role in secondary craniocerebral injury, leading to poor outcomes for TBI patients. Abrocitinib, a Janus kinase1 (JAK1) selective inhibitor approved to treat atopic dermatitis (AD) by the Food and Drug Administration (FDA), possesses a novel anti-inflammatory effect. In this study, we investigated whether abrocitinib could ameliorate neuroinflammation and exert a neuroprotective effect in traumatic brain injury (TBI) models., Methods: First, next-generation sequencing (NGS) was used to select genes closely related to neuroinflammation after TBI. Then, magnetic resonance imaging (MRI) was used to dynamically observe the changes in traumatic focus on the 1st, 3rd, and 7th days after the induction of fluid percussion injury (FPI). Moreover, abrocitinib's effects on neurobehaviors were evaluated. A routine peripheral blood test was carried out and Evans blue dye extravasation, cerebral cortical blood flow, the levels of inflammatory cytokines, and changes in the numbers of inflammatory cells were evaluated to investigate the function of abrocitinib on the 1st day post-injury. Furthermore, the JAK1/signal transducer and activator of transcription1 (STAT1)/nuclear factor kappa (NF-κB) pathway was assessed., Results: In vivo, abrocitinib treatment was found to shrink the trauma lesions. Compared to the TBI group, the abrocitinib treatment group showed better neurological function, less blood-brain barrier (BBB) leakage, improved intracranial blood flow, relieved inflammatory cell infiltration, and reduced levels of inflammatory cytokines. In vitro, abrocitinib treatment was shown to reduce the pro-inflammatory M1 microglia phenotype and shift microglial polarization toward the anti-inflammatory M2 phenotype. The WB and IHC results showed that abrocitinib played a neuroprotective role by restraining JAK1/STAT1/NF-κB levels after TBI., Conclusions: Collectively, abrocitinib treatment after TBI is accompanied by improvements in neurological function consistent with radiological, histopathological, and biochemical changes. Therefore, abrocitinib can indeed reduce excessive neuroinflammation by restraining the JAK1/STAT1/NF-κB pathway.

Neuroinflammation is an important mediator of secondary injury pathogenesis that exerts dual beneficial and detrimental effects on pathophysiology of the central nervous system (CNS) after traumatic brain injury (TBI). Fluvoxamine is a serotonin selective reuptake inhibitor (SSRI) and has been reported to have the anti-inflammatory properties. However, the mechanisms and therapeutic effects of fluvoxamine in neuroinflammation after TBI have not be defined. In this study, we showed that fluvoxamine inhibited peripheral immune cell infiltration and glia activation at 3 days in mice subjected to TBI. Fluvoxamine treatment promoted microglial/macrophage phenotypic transformation from pro-inflammatory M1-phenotype to anti-inflammatory M2-phenotype in in vivo and in vitro experiments. In addition, fluvoxamine treatment attenuated neuronal apoptosis, blood-brain barrier (BBB) disruption, cerebrovascular damage, and post-traumatic edema formation, thereby improving neurological function of mice subjected to TBI. These findings support the clinical evaluation of fluvoxamine as a neuroprotective therapy for TBI.

The incidence of repetitive mild traumatic brain injury (rmTBI), one of the main risk factors for predicting neurodegenerative disorders, is increasing; however, its underlying mechanism remains unclear. As suggested by several studies, ferroptosis is possibly related to TBI pathophysiology, but its effect on rmTBI is rarely studied. Mesenchymal stromal cells (MSCs), the most studied experimental cells in stem cell therapy, exert many beneficial effects on diseases of the central nervous system, yet evidence regarding the role of MSCs in ferroptosis and post-rmTBI neurodegeneration is unavailable. Our study showed that rmTBI resulted in time-dependent alterations in ferroptosis-related biomarker levels, such as abnormal iron metabolism, glutathione peroxidase (GPx) inactivation, decrease in GPx4 levels, and increase in lipid peroxidation. Furthermore, MSC treatment markedly decreased the aforementioned rmTBI-mediated alterations, neuronal damage, pathological protein deposition, and improved cognitive function compared with vehicle control. Similarly, liproxstatin-1, a ferroptosis inhibitor, showed similar effects. Collectively, based on the above observations, MSCs ameliorate cognitive impairment following rmTBI, partially via suppressing ferroptosis, which could be a therapeutic target for rmTBI., (© 2022. The Author(s).)

Prognosis evaluation is crucial for the effective management of patients with acute traumatic brain injury (TBI). However, there is still a lack of routinely available blood indicators for mortality risk in clinical practice. To investigate whether blood red cell distribution width to platelet count ratio (RPR) correlates with hospital mortality of TBI, clinical data of 2220 patients with TBI were extracted from two large intensive care unit cohorts (MIMIC-III and eICU Database), and were integratively analyzed using our developed method named MeDICS. We found that higher RPR can be observed among non-survivors than survivors of TBI ( p  < 0.001). It had a moderately good prognostic performance for mortality with an area under receiver-operating characteristic curve (AUC) of 0.7367, which was greater than that of Glasgow Coma Scale (GCS; AUC = 0.6022). Besides, the nomogram consisting of RPR, GCS, and other risk factors was developed, where 10-fold cross-validation was performed to protect it against overfitting. A Harrell's C-index of 0.8523 was determined, suggesting an improved prognostic value based on RPR. The in vivo experiments further confirmed the association between RPR and neuro-outcome after TBI. It indicated that the continuous change in RPR post-injury is attributed to the development of inflammation, which emphasized the importance of controlling inflammatory response in clinical treatment. Taken together, RPR is a promising routinely available predictor of mortality for acute TBI. The nomogram generated from it can be used in resource-limited settings, thus be proposed as a prognosis evaluation aid for patients with TBI in all levels of medical system.

In our recent study, we observed consistent increases in miR-124-3p levels in exosomes derived from cultured BV2 microglia which was treated with repetitive traumatic brain injury (rTBI) mouse model brain extracts. To clarify the mechanisms underlying increases in microglia-derived exosomal miR-124-3p and their role in regulating neuronal autophagy after TBI, we investigated the impact of exosomal miR-124-3p on neuronal autophagy in scratch-injured HT22 neurons and rTBI mice. We harvested injured brain extracts from rTBI mice at 3 to 21 days post injury (DPI) for the treatment of cultured BV2 microglia in vitro. We observed significant induction of autophagy following TBI in vitro, and that inhibition of activated neuronal autophagy could protect against trauma-induced injury. Our results indicated that co-culture of injured HT22 neurons with miR-124-3p overexpressing BV2 microglia exerted a protective effect by inhibiting neuronal autophagy in scratch-injured neurons. Further research revealed that these effects were achieved mainly via upregulation of exosomal miR-124-3p, and that Focal adhesion kinase family-interacting protein of 200 kDa (FIP200) plays a key role in trauma-induced autophagy. Injection of exosomes into the vena caudalis in in vivo experiments revealed that exosomal miR-124-3p was associated with decreases in the modified neurological severity score (mNSS) and improvements in Morris water maze (MWM) test results in rTBI mice. Altogether, our results indicate that increased miR-124-3p in microglial exosomes following TBI may inhibit neuronal autophagy and protect against nerve injury via their transfer into neurons. Thus, treatment with microglial exosomes enriched with miR-124-3p may represent a novel therapeutic strategy for the treatment of nerve injury after TBI.

Our recent articles have reported that increased miR-21-5p in brain after traumatic brain injury (TBI) could improve the neurological outcome through alleviating blood-brain barrier (BBB) damage. miR-21-3p is another mature miRNA derived from pre-miR-21 after Dicer Procession other than miR-21-5p. Its roles in various diseases, such as tumors and myocardial disease, aroused great interest for research in recent years. To further explore the function and underlying mechanism of miR-21, especially miR-21-3p, in regulating the pathological development of BBB damage after TBI, we designed this research and focused on studying the impact of miR-21-3p on apoptosis and inflammation in brain microvascular endothelial cells (BMVECs), the major cellular component of BBB. We performed controlled cortical impact on mouse brain and employed the oxygen glucose deprivation/reoxygenation (OGD)-treated bEnd.3 cells injury model. We found that the miR-21-3p level in BMVECs from injured cerebral cortex of controlled cortical impact (CCI) mice and bEnd.3 cells with OGD treatment were both increased after injury. For in vitro experiments, downregulation on the miR-21-3p level by transfecting miR-21-3p antagomir in cultured cells alleviated OGD-induced BBB damage, characterized by decreased BBB leakage and increased expression of tight junction proteins. Besides, miR-21-3p antagomir could suppress cell death by anti-apoptosis and control inflammatory response by inhibiting the activity of NF-κB signaling. Using luciferase reporter assay and a MAT2B-silenced shRNA vector, we further proved that miR-21-3p exerted the above functions through targeting MAT2B. In addition, in vivo experiments also confirmed that intracerebroventricular infusion of miR-21-3p antagomir could alleviate BBB leakage after TBI. It reduced Evans Blue extravasation and promoted the expression of tight junction proteins, thus contributed to improve the neurological outcome of CCI mice. Taken together, increased miR-21-3p in BMVECs after TBI was bad for restoration of injured BBB. Downregulation on the miR-21-3p level in injured brain could be a promising therapeutic strategy for BBB damage after TBI.

BACKGROUND Traumatic brain injury (TBI) produces a series of pathological processes. Recent studies have indicated that autophagy pathway is persistently activated after TBI, which may lead to deterioration of nerve injury. Our preliminary work found miR-21-5p was upregulated in both in vivo and in vitro TBI models. MicroRNAs (miRNAs) could be loaded into exosomes to perform cell-to-cell interactions. This research aimed to evaluate the therapeutic effect of neuron-derived exosomes enriched with miR-21-5p on the TBI in vitro and to further explore the possible mechanisms. MATERIAL AND METHODS Brain extracts harvested from an rTBI mouse model were added to cultured HT-22 neurons to imitate the microenvironment of injured brain on in vitro cultured cells. Ultracentrifugation was performed to isolate exosomes. Transmission electron microscopy and Nano sight technology were used to examine exosomes. An in vitro model of TBI was established to study the effect of exosomal miR-21-5p on nerve injury and on neuronal autophagy regulation. RESULTS The expression of miR-21-5p was increased in exosomes derived from HT-22 neurons after treatment with rTBI mouse brain extracts. Autophagy was activated in HT-22 neurons after scratch injury. Exosomal miR-21-5p produced a protective effect by suppressing autophagy in a TBI model in vitro. MiR-21-5p could directly target the Rab11a 3'UTR region to reduce its translation and further suppressed Rab11a-mediated neuronal autophagy. CONCLUSIONS The levels of miR-21-5p in neuronal exosomes increased from the acute to the chronic phase of TBI. Neuronal exosomes enriched with miR-21-5p can inhibit the activity of neuronal autophagy by targeting Rab11a, thus attenuating trauma-induced, autophagy-mediated nerve injury in vitro.

Pyroptosis is a highly specific type of inflammatory programmed cell death that different from necrosis or apoptosis. It is initiated by cellular detection of acute damage via recognizing pathogen-associated molecular patterns (PAMPs) by NOD-like receptors (NLRs) or AIM2-like receptor (AIM2). NLRs and AIM2 could trigger the formation of a multi-protein complex, known as inflammasome. It also contains apoptotic speck-containing protein (ASC) and pro-Caspase-1, and could process the signals to induce a cascade of inflammatory response. Recently, growing evidence showed that inflammasome-mediated pyroptosis is involved in the pathogenesis of traumatic brain injury (TBI). However, less attention has been paid to their particular roles in regulating blood-brain barrier (BBB) damage, the central pathological change in secondary brain damage of TBI. Thus, we designed this research to explore the impact and mechanism of NLRs and AIM2 inflammasome-mediated pyroptosis in BBB after TBI. We employed the controlled cortical impact (CCI) mice model and manipulated the severity of pyroptosis in BBB using Caspase-1 inhibitor, Ac-YVAD-cmk. We found that TBI led to NLRs and AIM2 inflammasome-mediated pyroptosis in brain microvascular endothelial cells (BMVECs) from injured cerebral cortex. Ac-YVAD-cmk treatment inhibited pyroptosis in injured BMVECs by suppressing the expression of essential inflammasome subunit - Caspase-1 and pivotal downstream pro-inflammatory cytokines (IL-1β and IL-18), as well as hindering GSDMD cleavage and ASC oligomerization. In addition, inhibiting pyroptosis could alleviate TBI-induced BBB leakage, brain edema, loss of tight junction proteins, and the inflammatory response in injured BMVECs. These effects contributed to improving the neurological outcome of CCI mice. In conclusion, NLRs and AIM2 inflammasome-mediated pyroptosis could aggravate BBB damage after TBI. Targeting and controlling pyroptosis in injured BBB would be a promising therapeutic strategy for TBI in the future., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuronal inflammation is the characteristic pathologic change of acute neurologic impairment and chronic traumatic encephalopathy after traumatic brain injury (TBI). Inhibiting the excessive inflammatory response is essential for improving the neurologic outcome. To clarify the regulatory mechanism of microglial exosomes on neuronal inflammation in TBI, we focused on studying the impact of microglial exosomal miRNAs on injured neurons in this research. We used a repetitive (r)TBI mouse model and harvested the injured brain extracts from the acute to the chronic phase of TBI to treat cultured BV2 microglia in vitro The microglial exosomes were collected for miRNA microarray analysis, which showed that the expression level of miR-124-3p increased most apparently in the miRNAs. We found that miR-124-3p promoted the anti-inflamed M2 polarization in microglia, and microglial exosomal miR-124-3p inhibited neuronal inflammation in scratch-injured neurons. Further, the mammalian target of rapamycin (mTOR) signaling was implicated as being involved in the regulation of miR-124-3p by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Using the mTOR activator MHY1485 we confirmed that the inhibitory effect of exosomal miR-124-3p on neuronal inflammation was exerted by suppressing the activity of mTOR signaling. PDE4B was predicted to be the target gene of miR-124-3p by pathway analysis. We proved that it was directly targeted by miR-124-3p with a luciferase reporter assay. Using a PDE4B overexpressed lentivirus transfection system, we suggested that miR-124-3p suppressed the activity of mTOR signaling mainly through inhibiting the expression of PDE4B. In addition, exosomal miR-124-3p promoted neurite outgrowth after scratch injury, characterized by an increase on the number of neurite branches and total neurite length, and a decreased expression on RhoA and neurodegenerative proteins [Aβ-peptide and p-Tau]. It also improved the neurologic outcome and inhibited neuroinflammation in mice with rTBI. Taken together, increased miR-124-3p in microglial exosomes after TBI can inhibit neuronal inflammation and contribute to neurite outgrowth via their transfer into neurons. miR-124-3p exerted these effects by targeting PDE4B, thus inhibiting the activity of mTOR signaling. Therefore, miR-124-3p could be a promising therapeutic target for interventions of neuronal inflammation after TBI. miRNAs manipulated microglial exosomes may provide a novel therapy for TBI and other neurologic diseases.-Huang, S., Ge, X., Yu, J., Han, Z., Yin, Z., Li, Y., Chen, F., Wang, H., Zhang, J., Lei, P. Increased miR-124-3p in microglial exosomes following traumatic brain injury inhibits neuronal inflammation and contributes to neurite outgrowth via their transfer into neurons., (© FASEB.)