Hyperglycemia disrupted the integrity of the blood-brain barrier following diffuse axonal injury through the sEH/NF-κB pathway.

OBJECTIVES: We aimed to investigate the role of soluble epoxide hydrolase for hyperglycemia induced-disruption of blood-brain barrier (BBB) integrity after diffuse axonal injury (DAI). METHODS: Rat DAI hyperglycemia model was established by a lateral head rotation device and intraperitoneal injection of 50% glucose. Glial fibrillary acidic protein, ionized calcium-binding adapter molecule-1, ß-amyloid precursor protein, neurofilament light chain, and neurofilament heavy chain was detected by immunohistochemistry. Cell apoptosis was examined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay. The permeability of blood-brain barrier (BBB) was assessed by expression of tight junction proteins, leakage of Evans blue and brain water content. The soluble epoxide hydrolase (sEH) pathway was inhibited by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) and the nuclear transcription factor kappa B (NF-κB) pathway was inhibited by pyrrolidine dithiocarbamate and activated by phorbol-12-myristate-13-acetate in vivo and/or vitro, respectively. The inflammatory factors were detected by enzyme-linked immunosorbent assay. RESULTS: Hyperglycemia could exacerbate axonal injury, aggravate cell apoptosis and glial activation, worsen the loss of BBB integrity, increase the release of inflammatory factors, and upregulate the expression of sEH and NF-κB. Inhibition of sEH could reverse all these damages and protect BBB integrity by upregulating the expression of tight junction proteins and downregulating the levels of inflammatory factors in vivo and vitro, while the agonist of NF-κB pathway abrogated the protective effects of TPPU on BBB integrity in vitro. CONCLUSIONS: sEH was involved in mediating axonal injury induced by hyperglycemia after DAI by disrupting BBB integrity through inducing inflammation via the NF-κB pathway.

Hyperglycemia Aggravates Blood-Brain Barrier Disruption Following Diffuse Axonal Injury by Increasing the Levels of Inflammatory Mediators through the PPARγ/Caveolin-1/TLR4 Pathway.

Hyperglycemia aggravates brain damage after diffuse axonal injury (DAI), but the underlying mechanisms are not fully defined. In this study, we aimed to investigate a possible role for hyperglycemia in the disruption of blood-brain barrier (BBB) integrity in a rat model of DAI and the underlying mechanisms. Accordingly, 50% glucose was intraperitoneally injected after DAI to establish the hyperglycemia model. Hyperglycemia treatment aggravated neurological impairment and axonal injury, increased cell apoptosis and glial activation, and promoted the release of inflammatory factors, including TNF-α, IL-1ß, and IL-6. It also exacerbated BBB disruption and decreased the expression of tight junction-associated proteins, including ZO-1, claudin-5, and occludin-1, whereas the PPARγ agonist rosiglitazone (RSG) had the opposite effects. An in vitro BBB model was established by a monolayer of human microvascular endothelial cells (HBMECs). Hyperglycemia induction worsened the loss of BBB integrity induced by oxygen and glucose deprivation (OGD) by increasing the release of inflammatory factors and decreasing the expression of tight junction-associated proteins. Hyperglycemia further reduced the expression of PPARγ and caveolin-1, which significantly decreased after DAI and OGD. Hyperglycemia also further increased the expression of toll-like receptor 4 (TLR4), which significantly increased after OGD. Subsequently, the PPARγ agonist RSG increased caveolin-1 expression and decreased TLR4 expression and inflammatory factor levels. In contrast, caveolin-1 siRNA abrogated the protective effects of RSG in the in vitro BBB model of hyperglycemia by increasing TLR4 and Myd88 expression and the levels of inflammatory factors, including TNF-α, IL-1ß, and IL-6. Collectively, we demonstrated that hyperglycemia was involved in mediating secondary injury after DAI by disrupting BBB integrity by inducing inflammation through the PPARγ/caveolin-1/TLR4 pathway.

NLRP3 inflammasome inhibitor MCC950 reduces cerebral ischemia/reperfusion induced neuronal ferroptosis.

The role of nucleotide-binding oligomerization domainlike receptor pyrin domain containing 3 (NLRP3) inflammasome in cerebral ischemia-reperfusion (I/R) induced neuroinflammation and neuronal pyroptosis has been widely recognized. Latest studies revealed that NLRP3 inflammasome engage in not only pyroptosis but also other types of cell death. Ferroptosis has been proved to be closely associated with cerebral I/R injury. In this study, our objectives were to verify the inhibitory effect of the NLRP3-specific inhibitor MCC950 on cerebral I/R-mediated neuronal pyroptosis, and to explore the regulation and possible mechanism of MCC950 on cerebral I/R-mediated neuronal ferroptosis. Our data showed that the NLRP3-specific inhibitor, MCC950, effectively reversed the I/R-mediated NLRP3 inflammasome activation and neuronal pyroptosis. Furthermore, we found that I/R increased iron concentrations and levels of malondialdehyde (MDA), downregulated glutathione peroxidase 4 (GPX4) expression, and upregulated long chain fatty acid-CoA ligase 4 (FACL4) and prostaglandin endoperoxide synthase 2 (PTGS2) expression. Interestingly, these changes were also reversed by the MCC950. Finally, in vitro, we found that MCC950 significantly reduced ROS levels in OGD/R treated HT22 cells. In conclusion, pharmaceutical inhibition of NLRP3 by MCC950 attenuates I/R-induced neuronal ferroptosis, possibly by reducing ROS accumulation.

Downregulation of Sepina3n Aggravated Blood-Brain Barrier Disruption after Traumatic Brain Injury by Activating Neutrophil Elastase in Mice.

Traumatic brain injury (TBI) is the leading cause of death in young adults and the main cause of mortality and disability across all ages worldwide. We previously analyzed the expression profile data of TBI models obtained from the Gene Expression Omnibus (GEO) database and found that the seripina3n mRNA was markedly upregulated in the acute phase of TBI in four mRNA expression profile data sets, indicating that serpina3n may be involved in the pathophysiological process of TBI. Therefore, we further investigated the biological role and molecular mechanism of serpina3n in traumatic brain injury in this study. As a result, the endogenous level of sepina3n was markedly elevated in the cortex around the contusion sit in mice at day 1 and day 3 after TBI. Inhibiting the expression of serpina3n caused aggravation of neutrophil elastase (NE) expression, BBB disruption, and neurological deficit. With the inactivation of NE, even if serpina3n was silenced, the disruption of the BBB was not further aggravated. In vitro experiments further proved that recombinant serpina3n dose-dependently inhibited the activity of recombinant NE. Based on the above, this study demonstrated that the endogenous level of sepina3n was significantly elevated in the cortex around the contusion sit after TBI in mice, which reduced the secondary blood-brain barrier disruption by inhibiting the activity of neutrophil elastase.

Inhibition of PRMT5 attenuates cerebral ischemia/reperfusion-Induced inflammation and pyroptosis through suppression of NF-κB/NLRP3 axis.

Protein methylation is a prevalent post-translational modification after cerebral ischemia. Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse the formation of methylated residues on histones and non-histone proteins. Accumulating evidence suggested that PRMT5 might play a carcinogenic role in various cancers. However, the role of PRMT5 in cerebral ischaemia/reperfusion (I/R) injury remains unclear. In this project, middle cerebral artery occlusion/reperfusion (MCAO/R) model in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) model in human neuroblastoma SH-SY5Y cells were utilized to mimic disease state of cerebral I/R. We found that expression of inflammatory-related factors [Interleukin (IL)-1ß and IL-6)] and pyroptotic-related factor [N-term cleaved Gasdermin-D (GSDMD-N)] were up-regulated in both MCAO/R mice and OGD/R SH-SY5Y cells. In addition, both in vivo and in vitro, PRMT5 was aberrantly upregulated during cerebral I/R. However, these alterations induced by I/R were blocked by PRMT5 inhibitor LLY-283, and enhanced by overexpression of PRMT5. Furthermore, rescue experiment proved that PRMT5 plays a pro-inflammatory and pro-pyroptotic role by activating nuclear factor kappa B (NF-κB)/nucleotide-binding oligomerization domainlike receptor pyrin domain containing 3 (NLRP3) axis. Finally, we observed that treatment of LLY-283 alleviated neurological deficits and reduced infarct volume in the MCAO/R mice. Taken together, PRMT5 may be a potential therapeutic target for cerebral I/R injury.

Sestrin2 protects against traumatic brain injury by reinforcing the activation of Nrf2 signaling.

Sestrin2 (SESN2) is stress-inducible protein that confers cytoprotective effects against various noxious stimuli. Accumulating evidence has documented that SESN2 has potent anti-apoptosis and anti-oxidative stress functions. However, whether it provides neuroprotection in traumatic brain injury (TBI) models remains unexplored. The purpose of this study was to explore the regulatory effect of SESN2 on TBI using in vivo and in vitro models. We found that TBI resulted in a marked induction of SESN2 in the cerebral cortex tissues of mice. SESN2 overexpression in the brain by in vivo gene transfer significantly decreased neurological deficit, brain edema, and neuronal apoptosis of mice with TBI. Moreover, the overexpression of SESN2 significantly decreased the oxidative stress induced by TBI in mice. In vitro studies of TBI demonstrated that SESN2 overexpression decreased apoptosis and oxidative stress in scratch-injured cortical neurons. Notably, SESN2 overexpression increased the nuclear levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and enhanced the activation of Nrf2 antioxidant signaling in in vivo and in vitro models of TBI. In addition, the inhibition of Nrf2 significantly abolished SESN2-mediated neuroprotective effects in vivo and in vitro. In conclusion, these results of our work demonstrate that SESN2 protects against TBI by enhancing the activation of Nrf2 antioxidant signaling.

Recombinant osteopontin provides protection for cerebral infarction by inhibiting the NLRP3 inflammasome in microglia.

Neuroinflammation is one of the most important secondary pathological events after cerebral infarction. Activation of NLRP3 inflammasome is a pivotal form of neuroinflammation. Osteopontin (OPN) is expressed during the subacute phase after cerebral infarction and has an important chemotactic effect on microglia. The aim of this study was to reveal the effect of recombinant OPN on brain injury after cerebral infarction and the regulation of NLRP3 inflammasome. We used the middle cerebral artery occlusion (MCAO) method-established focal cerebral ischemia model and LPS-induced inflammation model on neonate rat primary microglia. The effects of OPN on cerebral ischemic injury, neural function, microglia inflammation and NLRP3 inflammasome function were studied by immunofluorescence, staining, enzyme-linked immunosorbent assay and Western blot assay. We established MCAO cerebral ischemia and reperfusion injury model, and found that recombinant OPN reduced the volume of cerebral infarction and alleviated the ischemic injury degree of cerebral tissues, neurons, and neurological function. We found that OPN was also involved in the negative regulation of inflammasome and microglia activity in cerebral ischemic injury, and that OPN inhibited the activation of NLRP3 inflammasome and the function of microglia in a LPS-induced inflammatory model. Our findings show that recombinant OPN can reduce the ischemic infarct size and alleviate the cerebral ischemic injury of rats, which may be related to its efficient involvement in the inhibitory regulation of inflammasome and microglia inflammatory activation.

Inhibition of Macrophage Migration Inhibitory Factor Protects against Inflammation through a Toll-like Receptor-Related Pathway after Diffuse Axonal Injury in Rats.

OBJECTIVE: We have previously demonstrated that inflammation induced by toll-like receptors (TLRs) 2/4 exert cerebral deleterious effects after diffuse axonal injury (DAI); however, the underlying mechanisms are not fully understood. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine involved in inflammatory responses. The purpose of this study was to investigate the role of MIF in inflammation induced by TLRs in the cortices of DAI rats. METHODS: The rat DAI model was established by head rotational acceleration and confirmed by ß-APP, HE, and silver staining. MIF protein expression at 3 h, 6 h, 12 h, 1 d, and 3 d after DAI was measured by western blot. The localization of MIF was measured by immunofluorescence. MIF antagonist ISO-1 was intracerebroventricularly injected to inhibit MIF. Neuronal and axonal injury and glial responses were assessed by TUNEL, immunohistochemistry, and TEM. Expression of TLR2, TLR4, ERK, phospho-ERK, NF-κB, and phospho-NF-κB was examined by western blot. The level of IL-1ß, IL-6, and TNF-α was measured by ELISA. RESULTS: MIF expression was significantly increased, peaking at 1 day after DAI, and MIF was mainly localized in microglial cells and neurons. ISO-1 suppressed neuronal apoptosis, axonal injury, and glial responses and decreased the expression of downstream signaling molecules related to TLR2/4, including ERK, phospho-ERK, NF-κB, phospho-NF-κB, IL-1ß, IL-6, and TNF-α. CONCLUSION: MIF was involved in the neuronal and axonal damage through a TLR-related pathway following DAI.

High expression of EphA2 led to secondary injury by destruction of BBB integrity though the ROCK pathway after diffuse axonal injury.

Blood-brain barrier (BBB) disruption exacerbates diffuse axonal injury (DAI), but the underlying mechanisms are not fully understood. Inactivation or deletion of erythropoietin-producing hepatoma (EPH) receptor A2 (EphA2) attenuated BBB damage and promoted tight junction formation. In this study, we aimed to investigate the role of EphA2 in the protection of BBB integrity and the relevant mechanisms involved in a rat model of DAI. Blocking activation of the EphA receptor by EphA2-Fc ameliorated axonal injury, cell apoptosis, and glial activation, protected BBB integrity and increased expression of the tight junction-associated proteins ZO-1, claudin-5 and occludin-1. In vitro BBB models established by human brain microvascular endothelial cells (HBMECs) were subjected to oxygen deprivation (OGD). Treatment with EphrinA1, which activates EphA2, exacerbated the OGD-induced destruction of permeability and integrity of the BBB models by reducing the expression of tight junction-associated proteins. However, inhibition of Rho-associated coiled coil-containing protein kinases 1 and 2 (ROCK1 and 2) abrogated all of the effects of EphrinA1 on the BBB models in vitro. In conclusion, we provide evidence that EphA2 plays an important role in the destruction of BBB integrity by decreasing the expression of tight junction proteins through the ROCK pathway.

MCC950 Inhibits NLRP3 Inflammasome and Alleviates Axonal Injures in Early Stages of Diffuse Axonal Injury in Rats.

Increasing evidence has revealed that neuroinflammation plays a pivotal role in axonal injures. Nucleotide oligomerization domain (NOD)-like receptor protein (NLRP3) inflammasome is reported to be widely involved with the pathology of central nervous system disorders. But the role of NLRP3 in diffuse axonal injury (DAI) are rarely reported. The purpose of this study was to investigate the expression of NLRP3 after diffuse axonal injury and the role of NLRP3 in axonal injures. The lateral head rotation device was used to establish DAI model of rats. Immunohistochemical staining for ß-amyloid precursor protein and Bielschowsky silver staining were used to assess axonal injures and axonal loss. Terminal Deoxynucleotidyl Transferase-Mediated Digoxigenin-dUTP-Biotin Nick-End Labelling Assay was used to detect cell apoptosis. Brain water content was used to assess cerebral edema and the modified Neurologic Severity Score was used to assess the neurological deficits. Components of NLRP3 inflammasome, such as NLRP3, apoptosis-associated speck-like (ASC) adapter protein and caspase-1, and pro-inflammatory cytokines, for example IL-18 and IL-1ß, were over-expressed in early stages of DAI. MCC950, a selective small-molecule inhibitor of NLRP3 inflammasome, inhibited the over-expression of NLRP3 inflammasome and pro-inflammatory cytokines after DAI. MCC950 alleviated axonal injures and cell apoptosis. MCC950 also decreased brain water content and alleviated neurologic deficits 1 day and 3 days after DAI but not 7 days after DAI. These results suggest that MCC950 treatment in the early stages of DAI has a time limiting effect in preventing from axonal injuries and neurological deficits, and that NLRP3 inflammasome plays an important role in axonal injures and may be a potential candidate for axonal injures following DAI.

TGFß1 alleviates axonal injury by regulating microglia/macrophages alternative activation in traumatic brain injury.

Traumatic brain injury (TBI) causes substantial mortality and long-term disability worldwide. TGFß1 is a unique molecular and functional signature in microglia, but the role of TGFß1 in TBI is not clear. The purpose of this study was to investigate the role of TGFß1 in TBI. The weight dropping device was used to establish TBI model of rats. Hematoxylin eosin staining and Bielschowsky silver staining were used to assess tissue loss. Beam walking and muscle strength tests were used to assess neurological deficits. Immunohistochemical staining was used to assess axonal injures. Western blotting was used to detect expression of related proteins. RT-PCR was used to detect expression of cytokines. Immunofluorescence staining was used to assess the microglia/macrophages activation. We observed obvious axonal injury and microglia/macrophages activation in the peri-lesion cortex. The expression of inflammatory cytokines was markedly high after TBI. The expression of TGFß1 and TGFßRI were significantly reduced after TBI. TGFß1 promoted the functional recovery and alleviated axonal injury 1 day after TBI. TGFß1 promoted microglia/macrophages polarizing to alternative activation and alleviated neuroinflammation. These effects of TGFß1 could be inhibited by LY2109761, the inhibitor of TGFRI/II. These results suggested that TGFß1 played a protective role in axonal injury and could be a potential therapeutic target in early stages following TBI.

Effect and mechanism of YB-1 knockdown on glioma cell growth, migration, and apoptosis.

Y-box binding protein 1 (YB-1) is manifested as its involvement in cell proliferation and differentiation and malignant cell transformation. Overexpression of YB-1 is associated with glioma progression and patient survival. The aim of this study is to investigate the influence of YB-1 knockdown on glioma cell progression and reveal the mechanisms of YB-1 knockdown on glioma cell growth, migration, and apoptosis. It was found that the knockdown of YB-1 decreased the mRNA and protein levels of YB-1 in U251 glioma cells. The knockdown of YB-1 significantly inhibited cell proliferation, colony formation, and migration in vitro and tumor growth in vivo. Proteome and phosphoproteome data revealed that YB-1 is involved in glioma progression through regulating the expression and phosphorylation of major proteins involved in cell cycle, adhesion, and apoptosis. The main regulated proteins included CCNB1, CCNDBP1, CDK2, CDK3, ADGRG1, CDH-2, MMP14, AIFM1, HO-1, and BAX. Furthermore, it was also found that YB-1 knockdown is associated with the hypo-phosphorylation of ErbB, mTOR, HIF-1, cGMP-PKG, and insulin signaling pathways, and proteoglycans in cancer. Our findings indicated that YB-1 plays a key role in glioma progression in multiple ways, including regulating the expression and phosphorylation of major proteins associated with cell cycle, adhesion, and apoptosis.

Inhibiting endogenous tissue plasminogen activator enhanced neuronal apoptosis and axonal injury after traumatic brain injury.

Tissue plasminogen activator is usually used for the treatment of acute ischemic stroke, but the role of endogenous tissue plasminogen activator in traumatic brain injury has been rarely reported. A rat model of traumatic brain injury was established by weight-drop method. The tissue plasminogen activator inhibitor neuroserpin (5 µL, 0.25 mg/mL) was injected into the lateral ventricle. Neurological function was assessed by neurological severity score. Neuronal and axonal injuries were assessed by hematoxylin-eosin staining and Bielschowsky silver staining. Protein level of endogenous tissue plasminogen activator was analyzed by western blot assay. Apoptotic marker cleaved caspase-3, neuronal marker neurofilament light chain, astrocyte marker glial fibrillary acidic protein and microglial marker Iba-1 were analyzed by immunohistochemical staining. Apoptotic cell types were detected by immunofluorescence double labeling. Apoptotic cells in the damaged cortex were detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling staining. Degenerating neurons in the damaged cortex were detected by Fluoro-Jade B staining. Expression of tissue plasminogen activator was increased at 6 hours, and peaked at 3 days after traumatic brain injury. Neuronal apoptosis and axonal injury were detected after traumatic brain injury. Moreover, neuroserpin enhanced neuronal apoptosis, neuronal injury and axonal injury, and activated microglia and astrocytes. Neuroserpin further deteriorated neurobehavioral function in rats with traumatic brain injury. Our findings confirm that inhibition of endogenous tissue plasminogen activator aggravates neuronal apoptosis and axonal injury after traumatic brain injury, and activates microglia and astrocytes. This study was approved by the Biomedical Ethics Committee of Animal Experiments of Shaanxi Province of China in June 2015.

Evaluation of Genetic Variants in MIR3142HG in Susceptibility to and Prognosis of Glioma.

OBJECTIVES: Studies have demonstrated that genetic variants in the miRNA-coding genes might be associated with cancer susceptibility and survival. Here, we aimed to investigate the influence of MIR3142HG single-nucleotide polymorphisms on the individual's susceptibility to and patients' prognosis of glioma. MATERIALS AND METHODS: Six variants were genotyped by Agena MassARRAY iPLEX Gold assay among 529 glioma patients and 502 healthy controls. Association of MIR3142HG polymorphisms with the risk for and prognosis of glioma was analyzed by logistic regression analysis and Cox proportional hazards model, respectively. RESULTS: In the risk analysis, rs17057846 (odds ratio [OR]=1.93, P=0.047), rs2961920 (OR=1.53, P=0.019), and rs58747524 (OR=1.23, P=0.046) polymorphisms were associated with increased glioma risk, while rs7727115 (OR=0.76, P=0.030) and rs1582417 (female individuals, OR=0.49, P=0.017) variants were associated with decreased risk. In the survival analysis, rs1582417 polymorphism (hazard ratio=1.26, P=0.017) contributed to poorer prognosis overall. Rs17057846, rs1582417, and rs2431689 polymorphisms were associated with prognosis of astrocytoma, and rs1582417, rs17057846, and rs58747524 variants were associated with the survival rate in patients with low-grade glioma (I to II). CONCLUSION: Our study provided the first evidence for the impact of rs1582417, rs17057846, rs2431689, rs2961920, rs58747524, and rs7727115 polymorphisms in MIR3142HG on the susceptibility to and/or prognosis of glioma in the Chinese Han population.

A Precise, Controllable in vitro Model for Diffuse Axonal Injury Through Uniaxial Stretch Injury.

Regarding the determination of the biomechanical parameters in a reliable in vitro cell model for diffuse axonal injury (DAI), our study aimed to demonstrate connections between those parameters and secondary axotomy through examination of morphological alterations under a variety of traumatic conditions. An in vitro cell model for DAI was established in primary cultured mouse neurons by uniaxial mechanical stretching of non-myelinated axons under various traumatic conditions: strain (ε) = 5, 10, 20, and 50%; strain time (t) = 500, 100, and 20 ms; strain rate ranging between 0.1 and 25 s-1. Axonal real strains (strainaxon) were measured as 4.53 ± 0.27, 9.02 ± 0.91, 17.75 ± 1.65, and 41.8 ± 4.4%. Axonal real strain rates (SRaxon) ranged between 0.096 ± 0.0054 and 20.9 ± 2.2 s-1. Results showed there was no obvious abnormality of axons with a lower strain condition (strainaxon < 17.75 ± 1.65%) during the acute phase within 30 min after injury. In contrast, acute axonal degeneration (AAD) was observed in the axons following injury with a higher strain condition (SRaxon > 17.75 ± 1.65%). In addition, the incidence and degree of AAD were closely correlated with strain rate. Specifically, AAD occurred to all axons that were examined, when ε = 50% (strainaxon = 41.8 ± 4.4%) for 20 ms, while no spontaneous rupture was observed in those axons. Besides, the concentration of Ca2+ within the axonal process was significantly increased under such traumatic conditions. Moreover, the continuity of axon cytoskeleton was interrupted, eventually resulting in neuronal death during subacute stage following injury. In this study, we found that there is a minimum strain threshold for the occurrence of AAD in non-myelinated axons of primary cultured mouse neurons, which ranges between 9.02 ± 0.91 and 17.75 ± 1.65%. Basically, the severity of axonal secondary axotomy post DAI is strain rate dependent under a higher strain above the threshold. Hence, a reliable and reproducible in vitro cell model for DAI was established, when ε = 50% (strainaxon = 41.8 ± 4.4%) for 20 ms.

Simvastatin pretreatment ameliorates t-PA-induced hemorrhage transformation and MMP-9/TIMP-1 imbalance in thromboembolic cerebral ischemic rats.

Background: The use of thrombolysis with tissue-plasminogen activator (t-PA) in patients with acute ischemic stroke (AIS) is limited by increased levels of matrix metalloproteinase-9 (MMP-9) and by the increased risk of hemorrhagic transformation (HT). In this study, we investigated the effects of simvastatin pretreatment on t-PA-induced MMP-9/tissue inhibitor of metalloproteinase-1 (TIMP-1) imbalance and HT aggravation in a rat AIS model. Methods: The rat AIS model was established by autologous blood emboli. Two weeks before surgery, rats were pretreated with simvastatin (60 mg/kg/d), and three hours after surgery, t-PA (10 mg/kg) was administered. MMP-9 and TIMP-1 levels in the infarcted zone and plasma were evaluated by Western blot analysis and ELISA; the level of HT was quantified by determining the hemoglobin content. RhoA activation was determined to clarify the potential effect. Results: The results suggested that pretreatment with simvastatin suppressed the increase in t-PA-induced MMP-9 levels and neutralized the elevated MMP-9/TIMP-1 ratio, but had no effect on TIMP-1 levels. Thrombolysis with t-PA after ischemia improved neurological outcome, but increased intracranial hemorrhage. Moreover, t-PA-induced HT aggravation was reduced by simvastatin pretreatment. In addition, we showed that t-PA-induced activation of RhoA was suppressed by simvastatin, and that t-PA-induced MMP-9/TIMP-1 imbalance and hemorrhage was reduced by Rho kinases (ROCK) inhibitor Y-27632. Conclusion: In this study, we showed that simvastatin pretreatment ameliorated t-PA-induced HT and MMP-9/TIMP-1 imbalance, and demonstrated that the RhoA/ROCK pathway was implicated.

LncRNA SNHG6 functions as a ceRNA to regulate neuronal cell apoptosis by modulating miR-181c-5p/BIM signalling in ischaemic stroke.

Long non-coding RNAs (lncRNAs) play important roles in the pathogenesis of brain and neurodegenerative disorders. As far as we know, the functions and potential mechanisms of small nucleolar RNA host gene 6 (SNHG6) in ischaemic stroke have not been explored. This study aimed to examine the functional role of SNHG6 in the ischaemic stroke. Middle cerebral artery occlusion (MCAO) in mice and the oxygen glucose deprivation (OGD)-induced injury in neuronal cells were applied to mimic ischaemic stroke. TTC staining, quantitative real-time PCR, cell apoptosis assay, caspase-3 activity assay, Western blot, RNA immunoprecipitation and luciferase reporter assay were performed to evaluate the function and possible mechanisms of SNHG6 in the pathogenesis of ischaemic stroke. The results show that SNHG6 expression was significantly increased both OGD-induced neuronal cells and MCAO model mice. In vitro results showed that inhibition of SNHG6 increased cell viability, inhibited cell apoptosis and caspase-3 activity in OGD-induced neuronal cells. Consistently, knockdown of SNHG6 reduced brain infarct size and improved neurological scores in the MCAO mice. Mechanistic study further revealed that SNHG6 functioned as a competing endogenous RNA (ceRNA) for miR-181c-5p, which in turn repressed its downstream target of Bcl-2 interacting mediator of cell death (BIM) and inhibiting cell apoptosis. This study revealed a novel function of SNHG6 in the modulating neuronal apoptosis in the ischaemic stroke model, and the role of SNHG6 in the regulating of neuronal apoptosis was at least partly via targeting miR-181c-5p/BIM signalling pathway.

Vascular endothelial growth factor A promotes platelet adhesion to collagen IV and causes early brain injury after subarachnoid hemorrhage.

The role of vascular endothelial growth factor A in platelet adhesion in cerebral microvessels in the early stage of subarachnoid hemorrhage remains unclear. In this study, the endovascular puncture method was used to produce a rat model of subarachnoid hemorrhage. Then, 30 minutes later, vascular endothelial growth factor A antagonist anti-vascular endothelial growth factor receptor 2 antibody, 10 µg, was injected into the right ventricle. Immunohistochemistry and western blot assay were used to assess expression of vascular endothelial growth factor A, occludin and claudin-5. Immunohistochemical double labeling was conducted to examine co-expression of GP Ia-II integrin and type IV collagen. TUNEL was used to detect apoptosis in the hippocampus. Neurological score was used to assess behavioral performance. After subarachnoid hemorrhage, the expression of vascular endothelial growth factor A increased in the hippocampus, while occludin and claudin-5 expression levels decreased. Co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells increased, whereas behavioral performance was markedly impaired. After treatment with anti-vascular endothelial growth factor receptor 2 antibody, occludin and claudin-5 expression recovered, while co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells decreased. Furthermore, behavioral performance improved notably. Our findings suggest that increased vascular endothelial growth factor A levels promote platelet adhesion and contribute to early brain injury after subarachnoid hemorrhage. This study was approved by the Biomedical Ethics Committee, Medical College of Xi'an Jiaotong University, China in December 2015.

Glial response in early stages of traumatic brain injury.

Traumatic brain in jury affects a number of individuals per year and is a major cause of worldwide death and disability. Yet, its pathophysiological mechanism remains unclear. It is well-known that glial cells, including microglia and astrocytes, are activated and involved in tissue damage and repair in the peri-lesion regions after traumatic brain injury; however, global glial responses are rarely reported. The purpose of this study was to investigate the global activation of microglia and astrocytes 1 day after traumatic brain injury. To test this, we used a weight drop device to inflict traumatic brain injury on left side of the brain and performed hematoxylin-eosin staining to detect tissue damage. We used immunohistochemical staining and western blotting to detect the activation of microglia and astrocytes 1 day after TBI. We found that microglia were significantly activated in ipsilateral regions. Interestingly, we found that astrocytes were also significantly activated in the ipsilateral regions, contralateral cortex, and contralateral corpus callosum. These results suggest that a focal damage can cause a global glial reaction.

Peroxisome Proliferator-Activated Receptor γ Agonist Rosiglitazone Protects Blood-Brain Barrier Integrity Following Diffuse Axonal Injury by Decreasing the Levels of Inflammatory Mediators Through a Caveolin-1-Dependent Pathway.

Our early experiments confirmed that rosiglitazone (RSG), a peroxisome proliferator-activated receptor γ (PPARγ) agonist, had therapeutic potential for the treatment of diffuse axonal injury (DAI) by inhibiting the expression of amyloid-beta precursor protein and reducing the loss and abnormal phosphorylation of tau, but the underlying mechanisms were not fully defined. In this study, we aimed to investigate a possible role for PPARγ in the protection of blood-brain barrier (BBB) integrity in a rat model of DAI, and the underlying mechanisms. PPAR agonists and antagonists were intraperitoneally injected after DAI. Treatment with RSG ameliorated axonal injury, cell apoptosis, glia activation, and the release of inflammatory factors such as TNF-α, IL-1ß, and IL-6. It also increased the expression of tight junction-associated proteins like ZO-1, claudin-5, and occludin-1, whereas the PPARγ antagonist GW9662 had the opposite effects. These effects were also studied in a BBB in vitro model, consisting of a monolayer of human microvascular endothelial cells (HBMECs) subjected to oxygen and glucose deprivation (OGD). Treatment with RSG ameliorated the loss of BBB integrity and the increased permeability induced by OGD by reducing the release of inflammatory factors and maintaining the expression of tight junction-associated proteins. Interestingly, caveolin-1 was found located mainly in endothelial cells, and RSG increased the expression of caveolin-1, which decreased following OGD. In contrast, caveolin-1 siRNA abrogated the protective effects of RSG in the in vitro BBB model. In conclusion, we provide evidence that PPARγ plays an important role in a series of processes associated with DAI, and that the PPARγ agonist RSG can protect BBB integrity by decreasing the levels of inflammatory mediators through a caveolin-1-dependent pathway.