LncRNA xist regulates sepsis associated neuroinflammation in the periventricular white matter of CLP rats by miR-122-5p/PKCη Axis.

Background: Neuroinflammation is a common feature of many neurological diseases, and remains crucial for disease progression and prognosis. Activation of microglia and astrocytes can lead to neuroinflammation. However, little is known about the role of lncRNA xist and miR-122-5p in the pathogenesis of sepsis-associated neuroinflammation (SAN). This study aims to investigate the role of lncRNA xist and miR-122-5p in the pathogenesis of SAN. Methods: Levels of miR-122-5p and proinflammatory mediators were detected in the cerebrospinal fluid (CSF) of patients with intracranial infection (ICI) by ELISA and qRT-PCR. miRNA expression in the periventricular white matter (PWM) in rats was analyzed by high-throughput sequencing. Levels of lncRNA xist, miR-122-5p and proinflammatory mediators in the PWM were measured using qRT-PCR and western blot. Bioinformatics analysis was used to predict the upstream and downstream of miR-122-5p. The interaction between miR-122-5p and its target protein was validated using luciferase reporter assay. BV2 and astrocytes were used to detect the expression of lncRNA xist, miR-122-5p. Results: The level of miR-122-5p was significantly decreased in the CSF of ICI patients, while the expression of IL-1ß and TNF-α were significantly upregulated. Furthermore, it was found that the expression of IL-1ß and TNF-α were negatively correlated with the level of miR-122-5p. A high-throughput sequencing analysis showed that miR-122-5p expression was downregulated with 1.5-fold changes in the PWM of CLP rats compared with sham group. Bioinformatics analysis found that lncRNA xist and PKCη were the upstream and downstream target genes of miR-122-5p, respectively. The identified lncRNA xist and PKCη were significantly increased in the PWM of CLP rats. Overexpression of miR-122-5p or knockdown of lncRNA xist could significantly downregulate the level of PKCη and proinflammatory mediators from activated microglia and astrocytes. Meanwhile, in vitro investigation showed that silencing lncRNA xist or PKCη or enhancing the expression of miR-122-5p could obviously inhibit the release of proinflammatory mediators in activated BV2 cells and astrocytes. Conclusion: LncRNA xist could regulate microglia and astrocytes activation in the PWM of CLP rats via miR-122-5p/PKCη axis, further mediating sepsis associated neuroinflammation.

MiRNA-338-3p Inhibits Neuroinflammation in the Corpus Callosum of LCV-LPS Rats Via STAT1 Signal Pathway.

Neuroinflammation is a common characteristic of intracranial infection (ICI), which is associated with the activation of astrocytes and microglia. MiRNAs are involved in the process of neuroinflammation. This study aimed to investigate the potential mechanism by which miR-338-3p negatively modulate the occurrence of neuroinflammation. We here reported that the decreased levels of miR-338-3p were detected using qRT-PCR and the upregulated expression of TNF-α and IL-1ß was measured by ELISA in the cerebrospinal fluid (CSF) in patients with ICI. A negative association between miR-338-3p and TNF-α or IL-1ß was revealed by Pearson correlation analysis. Sprague-Dawley (SD) rats were injected with LPS (50 µg) into left cerebral ventricule (LCV), following which the increased expression of TNF-α and IL-1ß and the reduction of miR-338-3p expression were observed in the corpus callosum (CC). Moreover, the expression of TNF-α and IL-1ß in the astrocytes and microglia in the CC of LCV-LPS rats were saliently inhibited by the overexpression of miR-338-3p. In vitro, cultured astrocytes and BV2 cells transfected with mimic-miR-338-3p produced less TNF-α and IL-1ß after LPS administration. Direct interaction between miR-338-3p and STAT1 mRNA was validated by biological information analysis and dual luciferase assay. Furthermore, STAT1 pathway was found to be implicated in inhibition of neuroinflammation induced by mimic miR-338-3p in the astrocytes and BV2 cells. Taken together, our results suggest that miR-338-3p suppress the generation of proinflammatory mediators in astrocyte and BV2 cells induced by LPS exposure through the STAT1 signal pathway. MiR-338-3p could act as a potential therapeutic strategy to reduce the neuroinflammatory response. Diagram describing the cellular and molecular mechanisms associated with LPS-induced neuroinflammation via the miR-338-3p/STAT1 pathway. LPS binds to TLRs on astrocytes or microglia to activate the STAT1 pathway and upregulate the production of pro-inflammatory cytokines. However, miR-338-3p inhibits the expression of STAT1 and reduces the production of inflammatory mediators.

Influence of Melatonin on Behavioral and Neurological Function of Rats with Focal Cerebral Ischemia-Reperfusion Injury via the JNK/FoxO3a/Bim Pathway.

OBJECTIVE: To investigate the influence of melatonin on behavioral and neurological function of rats with focal cerebral ischemia-reperfusion injury via the JNK/FoxO3a/Bim pathway. METHODS: One hundred and twenty healthy male SD rats were randomized into the model group (Model: the middle cerebral artery occlusion (MCAO) model was constructed and received an equal volume of normal saline containing 5% DMSO), sham operation group (Sham: received no treatment except normal feeding), and low, medium, and high dose of melatonin group (L-MT, M-MT, and H-MT intraperitoneally injected 10, 20, and 40 mg/kg melatonin 30 min after IR, respectively), with 24 rats in each group. Following 24 h of reperfusion, the rats in each of the above groups were tested for neurological deficit symptoms and behavioral changes to screen the rats included in the study. HE and TUNEL stainings were performed to observe pathological changes. Levels of oxidative stress-related indexes, inflammatory factor-related indexes, nuclear factor-κB p65 (NF-κB p65), and interferon-γ (IFN-γ) in the rat brain were measured by ELISA. The JNK/FoxO3a/Bim pathway-related proteins as well as Bcl-2, Caspase-3, and Bax were examined using Western blot. RESULTS: Detection of behavioral indicators showed that the MACO model was successfully constructed in rats. L-MT, M-MT, and L-MT groups presented reduced malondialdehyde (MDA), reactive oxygen species (ROS), tumor necrosis factor- (TNF-) α, interleukin- (IL-) 6, IL-1ß, IFN-γ, NF-κB p65, and apoptosis compared with the Model group (P < 0.05), and the improvement degree was better in the M-MT group versus the L-HT group. Bcl-2 protein expression in the brain tissue of L-MT, M-MT, and H-MT groups increased significantly, while Bax, Caspase-3, p-JNK, p-FoxO3a, and Bim protein expression declined markedly, versus the Model group (P < 0.05). The changes of indexes were greater in the M-MT group compared with that in the L-MT group. No significant difference was observed in all the above indexes between the M-MT group and the H-MT group (P > 0.05). CONCLUSIONS: In the MACO rat model, melatonin can effectively reduce Bax and Caspase-3 levels by modulating the JNK/FoxO3a/Bim pathway, inhibit neuronal apoptosis, and alleviate neurological deficits by reducing the release of proinflammatory mediators, with anti-inflammatory and antioxidant effects. In addition, 20 mg/kg is the optimal melatonin concentration.

Extracellular vesicles derived from lung cancer cells exposed to intermittent hypoxia upregulate programmed death ligand 1 expression in macrophages.

PURPOSE: Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), compromises immune surveillance through the upregulation of programmed cell death-1 ligand (PD-L1). Tumor-released extracellular vesicles (EVs) have been reported to modulate immunosuppressive activities. We investigated whether or not EVs derived from intermittent hypoxic lung cancer cells can alter the expression of PD-L1 in macrophages. METHODS: The expression of PD-L1+monocytes from 40 patients with newly diagnosed non-small-cell lung cancer (NSCLC) and with (n=21) or without (n=19) OSA were detected. Plasma EVs isolated from NSCLC patients with moderate-severe OSA (n=4) and without OSA (n=4) were co-cultured with macrophages. A549 cells were exposed to normoxia or IH (48 cycles of 5 min of 1% O2 hypoxia, followed by 5 min of normoxia). EVs were isolated from cell supernatant and were co-cultured with macrophages differentiated from THP-1. PD-L1 and hypoxia-inducible factor-1 α (HIF-1α) expressions were measured by flow cytometry, immunofluorescence, and Western blot analysis. RESULTS: PD-L1+monocytes were elevated in NSCLC patients with OSA and increased with the severity of OSA and nocturnal desaturation. PD-L1+ macrophages were induced by EVs from NSCLC patients with OSA and positively correlated with HIF-1α expressions. EVs from IH-treated A549 can promote PD-L1 and HIF-1α expression in macrophages and the upregulation of PD-L1 expression was reversed by specific HIF-1α inhibitor. CONCLUSION: IH can enhance the function of EVs derived from lung cancer cells to aggravate immunosuppressive status in macrophages. HIF-1α may play an important role in this process.

A nomogram incorporating functional and tubular damage biomarkers to predict the risk of acute kidney injury for septic patients.

BACKGROUND: Combining tubular damage and functional biomarkers may improve prediction precision of acute kidney injury (AKI). Serum cystatin C (sCysC) represents functional damage of kidney, while urinary N-acetyl-ß-D-glucosaminidase (uNAG) is considered as a tubular damage biomarker. So far, there is no nomogram containing this combination to predict AKI in septic cohort. We aimed to compare the performance of AKI prediction models with or without incorporating these two biomarkers and develop an effective nomogram for septic patients in intensive care unit (ICU). METHODS: This was a prospective study conducted in the mixed medical-surgical ICU of a tertiary care hospital. Adults with sepsis were enrolled. The patients were divided into development and validation cohorts in chronological order of ICU admission. A logistic regression model for AKI prediction was first constructed in the development cohort. The contribution of the biomarkers (sCysC, uNAG) to this model for AKI prediction was assessed with the area under the receiver operator characteristic curve (AUC), continuous net reclassification index (cNRI), and incremental discrimination improvement (IDI). Then nomogram was established based on the model with the best performance. This nomogram was validated in the validation cohort in terms of discrimination and calibration. The decision curve analysis (DCA) was performed to evaluate the nomogram's clinical utility. RESULTS: Of 358 enrolled patients, 232 were in the development cohort (69 AKI), while 126 in the validation cohort (52 AKI). The first clinical model included the APACHE II score, serum creatinine, and vasopressor used at ICU admission. Adding sCysC and uNAG to this model improved the AUC to 0.831. Furthermore, incorporating them significantly improved risk reclassification over the predictive model alone, with cNRI (0.575) and IDI (0.085). A nomogram was then established based on the new model including sCysC and uNAG. Application of this nomogram in the validation cohort yielded fair discrimination with an AUC of 0.784 and good calibration. The DCA revealed good clinical utility of this nomogram. CONCLUSIONS: A nomogram that incorporates functional marker (sCysC) and tubular damage marker (uNAG), together with routine clinical factors may be a useful prognostic tool for individualized prediction of AKI in septic patients.

Hypertonic saline mediates the NLRP3/IL-1ß signaling axis in microglia to alleviate ischemic blood-brain barrier permeability by downregulating astrocyte-derived VEGF in rats.

INTRODUCTION: The aim of this study was to explore whether the antibrain edema of hypertonic saline (HS) is associated with alleviating ischemic blood-brain barrier (BBB) permeability by downregulating astrocyte-derived vascular endothelial growth factor (VEGF), which is mediated by microglia-derived NOD-like receptor protein 3 (NLRP3) inflammasome. METHODS: The infarct volume and BBB permeability were detected. The protein expression level of VEGF in astrocytes in a transient focal brain ischemia model of rats was evaluated after 10% HS treatment. Changes in the NLRP3 inflammasome, IL-1ß protein expression, and the interleukin-1 receptor (IL1R1)/pNF-кBp65/VEGF signaling pathway were determined in astrocytes. RESULTS: HS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulates IL-1ß expression by inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulates VEGF expression by inhibiting the phosphorylation of NF-кBp65 mediated by IL-1ß in astrocytes. CONCLUSIONS: HS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulated IL-1ß expression via inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulated VEGF expression through inhibiting the phosphorylation of NF-кBp65 mediated by IL-1ß in astrocytes.

Hypertonic saline downregulates endothelial cell-derived VEGF expression and reduces blood-brain barrier permeability induced by cerebral ischaemia via the VEGFR2/eNOS pathway.

The aim of the present study was to explore the possible mechanisms by which hypertonic saline (HS) effectively ameliorates cerebral oedema via the vascular endothelial growth factor receptor 2 (VEGFR2)­mediated endothelial nitric oxide synthase (eNOS) pathway of endothelial cells in rats. A middle cerebral artery occlusion (MCAO) model in Sprague­Dawley rats and an oxygen­glucose deprivation (OGD) model in cells were used in the present study. Evans blue (EB) staining and a horseradish peroxidase flux assay were performed to evaluate the protective effect of 10% HS on the blood­brain barrier (BBB). The expression levels of vascular endothelial growth factor (VEGF), VEGFR2, zonula occludens 1 (ZO1) and occludin were quantified. The results demonstrated that 10% HS effectively reduced EB extravasation in the peri­ischaemic brain tissue. At 24 h after MCAO, the protein expression levels of VEGF and VEGFR2 in the peri­ischaemic brain tissue were downregulated following treatment with 10% HS. In vitro experiments demonstrated that the permeability of a monolayer endothelial cell barrier was decreased significantly following HS treatment. In addition, VEGF and VEGFR2 protein expression levels were increased in endothelial cells under hypoxic conditions, but that effect was suppressed by HS treatment. Furthermore, HS inhibited the downregulation of ZO1 and occludin effectively, possibly through the VEGFR2/phospholipase C γ1 (PLCγ1)/eNOS signalling pathway. In conclusion, 10% HS may alleviate cerebral oedema through reducing ischaemia­induced BBB permeability, as a consequence of inhibiting VEGFR2/PLCγ1/eNOS­mediated downregulation of ZO1 and occludin.

Synaptic structure and alterations in the hippocampus in neonatal rats exposed to lipopolysaccharide.

Synaptic structure integrity plays a key role in learning and memory. Previous studies have shown that there is cognitive dysfunction in septic neonates in later life. In this study, intraperitoneal injection of lipopolysaccharide (LPS) in the developing rats was used as a sepsis model to determine whether hippocampal synapses would be affected. Expression of synaptophysin (Syn), synaptosomal associated protein of 25 kD (SNAP-25), and N-methyl d-aspartate receptor (NMDAR) in the hippocampus in septic brain were significantly reduced. Consistent with this, the number of dendritic spines associated with the pyramidal neurons in the CA1 region of hippocampus at 28d after LPS administration was decreased. Additionally, the number of synapse and synaptic vesicles were reduced and appeared swollen. The number of neurons in the CA1 and CA3 of hippocampus at 14, and 28d after LPS injection remained unchanged. Coupled with the above was upregulated expression of interleukin-1ß (IL-1ß), IL-1 receptor 1 (IL-R1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) at 1-3d after LPS injection. IL-1ß expression was specifically detected in activated microglia. The plasma corticosterone (CORT) concentration in the LPS treatment rats was increased; but the glucocorticoid receptor (GR) expression in the hippocampus was decreased. We conclude that LPS injection in neonatal rats can cause synaptic disruption in the hippocampus which may be attributed to inflammatory response due to excess production of proinflammatory cytokines e.g., IL-1ß derived from activated microglia. The significance of increased plasma CORT concentration and decreased GR expression in the hippocampus is discussed.

Long non­coding RNA DILC is involved in sepsis by modulating the signaling pathway of the interleukin­6/signal transducer and activator of transcription 3/Toll­like receptor 4 axis.

Sepsis is characterized by systemic inflammatory responses. In the present study, the role of deleted in liver cancer 1 (DILC), interleukin (IL)­6, signal transducer and activator of transcription 3 (STAT3), and Toll­like receptor 4 (TLR4) in the pathogenesis of sepsis was investigated. Reverse transcription­quantitative polymerase chain reaction analysis and western blotting were performed to evaluate the effects of lipopolysaccharide (LPS) on the expression of DILC, IL­6, STAT3, and TLR4, in addition to the effects of DILC and IL­6 on the synthesis of tumor necrosis factor (TNF­α), chemokine ligand 5 (CCL5), E­selectin and C­X­C motif chemokine receptor 1 (CXCR1). In addition, the regulatory association between DILC, IL­6, STAT3 and TLR4 was investigated. LPS reduced the expression level of DILC, and enhanced the expression of IL­6, STAT3 and TLR4. DILC directly and negatively regulated the synthesis of IL­6, as demonstrated by the markedly decreased luciferase activity in cells transfected with a wild­type DILC plasmid. On the other hand, compared with the scramble control, DILC and IL­6 small interfering (si)RNAs significantly suppressed the expression of IL­6, STAT3 and TLR4. In addition, DILC siRNA enhanced the expression of IL­6, STAT3 and TLR4, whereas the expression levels of TNF­α, CCL5, E­selectin and CXCR1 in LPS­treated THP­1 cells were downregulated following transfection with DILC and IL­6 siRNAs. In patients with sepsis, DILC expression was inhibited, although the expression levels of IL­6, STAT3 and TLR4 were upregulated. In addition, the expression levels of TNF­α, CCL5, E­selectin and CXCR1 in patients with sepsis were higher compared with normal subjects. Therefore, DILC may mediate the crosstalk between the cascades of IL­6/STAT3 and TNF­α signaling, indicating that DILC may act as a prognostic biomarker of sepsis, and may serve as a potential therapeutic target for the treatment of sepsis.

Ulinastatin Ameliorates Pulmonary Capillary Endothelial Permeability Induced by Sepsis Through Protection of Tight Junctions via Inhibition of TNF-α and Related Pathways.

Background: Increased permeability of pulmonary capillary is a common consequence of sepsis that leads to acute lung injury. In this connection, ulinastatin, a urinary trypsin inhibitor (UTI), is used clinically to mitigate pulmonary edema caused by sepsis. However, the underlying mechanism of UTI in alleviating sepsis-associated pulmonary edema remains to be fully elucidated. As tight junctions (TJs) between the pulmonary microvascular endothelial cells (PMVECs) play a pivotal role in the permeability of pulmonary capillary, this study investigated the effect of UTI on expression of junctional proteins in PMVECs during sepsis. Methods: Male adult Sprague Dawley rats were subjected to cecal ligation and puncture (CLP) and divided into sham, CLP, and UTI+CLP groups. UTI was administered every 8 h for 3 days before CLP. At 48 h after surgery, Evans blue (EB) was administered to evaluate the pulmonary vascular leakage. Histological staining was used for evaluation of lung injury score. Using immunofluorescence staining and Western blot, the expression of junctional proteins (occludin, claudin-5, and ZO-1) in pulmonary endothelia was assessed. In vitro, PMVECs were divided into control, lipopolysaccharide (LPS), and UTI+LPS groups for examination of expression of junctional proteins and TNF-α as well as inhibitor of NF-κB (IκB), p38 mitogen-activated protein kinases (p38 MAPKs), c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) signaling pathways. Additionally, the expression of various junctional proteins was determined in PMVECs of control, LPS, and TNF-α receptor antagonist-LPS groups. PMVECs were also treated with TNF-α and TNF-α receptor antagonist and the expression of various junctional proteins was assessed. Results: Compared with the CLP group, UTI markedly decreased EB leakage and lung injury score. The expression of occludin, claudin-5, and ZO-1 was decreased in both CLP rats and LPS-treated PMVECs, but it was reversed by UTI and TNF-α receptor antagonist. TNF-α expression was vigorously elevated in the lung of CLP rats and in LPS-challenged PMVECs, which were suppressed by UTI. In addition, TNF-α also reduced occludin, claudin-5, and ZO-1 expression in PMVECs, but these effects of TNF-α were antagonized by pretreatment with TNF-α receptor antagonist. Furthermore, UTI inhibited LPS-induced activation of NF-κB and mitogen-activated protein kinases (MAPKs) pathways in PMVECs. Conclusion: UTI effectively protects TJs and helps to attenuate the permeability of pulmonary capillary endothelial cells during sepsis through inhibiting NF-κB and MAPKs signal pathways and TNF-α expression.

The Incidence, Risk Factors and Outcomes of Postoperative Acute Kidney Injury in Neurosurgical Critically Ill Patients.

We investigated the incidence, perioperative risk factors, and outcomes of postoperative acute kidney injury (AKI) in neurosurgical critically ill patients. A prospective multicenter cohort study was conducted, enrolling adult patients who underwent neurosurgical procedure and admitted to the neurosurgical intensive care units (ICU). Postoperative AKI was diagnosed within 7 days after surgery based on the Kidney Disease Improving Global Outcomes criteria. Of 624 enrolled patients, postoperative AKI occurred in 84 patients. AKI was associated with increased rates of ICU and in-hospital mortality, postoperative renal replacement therapy, postoperative tracheotomy, and postoperative tracheal reintubation. Patients who developed AKI had higher total ICU costs, prolonged length of hospital and ICU stay, and longer duration of postoperative mechanical ventilation. Multivariate analysis identified postoperative reoperation (adjusted odds ratio [OR] 5.70 [95% CI, 1.61-20.14]), postoperative concentration of serum cystatin C (adjusted OR 4.53 [95% CI, 1.98-10.39]), use of mannitol during operation (adjusted OR 1.97 [95% CI, 1.13-3.43]), postoperative APACHE II score (adjusted OR 1.11 [95% CI, 1.06-1.16]), and intraoperative estimated blood loss (adjusted OR 1.04 [95% CI, 1.00-1.08]) as independent risk factors for postoperative AKI. Postoperative AKI in neurosurgical critically ill cohort is prevalent and associated with adverse in-hospital outcomes.

Hypertonic saline attenuates expression of Notch signaling and proinflammatory mediators in activated microglia in experimentally induced cerebral ischemia and hypoxic BV-2 microglia.

BACKGROUND: Ischemic stroke is a major disease that threatens human health in ageing population. Increasing evidence has shown that neuroinflammatory mediators play crucial roles in the pathophysiology of cerebral ischemia injury. Notch signaling is recognized as the cell fate signaling but recent evidence indicates that it may be involved in the inflammatory response in activated microglia in cerebral ischemia. Previous report in our group demonstrated hypertonic saline (HS) could reduce the release of interleukin-1 beta and tumor necrosis factor-alpha in activated microglia, but the underlying molecular and cellular mechanisms have remained uncertain. This study was aimed to explore whether HS would partake in regulating production of proinflammatory mediators through Notch signaling. RESULTS: HS markedly attenuated the expression of Notch-1, NICD, RBP-JK and Hes-1 in activated microglia both in vivo and in vitro. Remarkably, HS also reduced the expression of iNOS in vivo, while the in vitro levels of inflammatory mediators Phos-NF-κB, iNOS and ROS were reduced by HS as well. CONCLUSION: Our results suggest that HS may suppress of inflammatory mediators following ischemia/hypoxic through the Notch signaling which operates synergistically with NF-κB pathway in activated microglia. Our study has provided the morphological and biochemical evidence that HS can attenuate inflammation reaction and can be neuroprotective in cerebral ischemia, thus supporting the use of hypertonic saline by clinicians in patients with an ischemia stroke.

Hypertonic saline protects brain endothelial cells against hypoxia correlated to the levels of epidermal growth factor receptor and interleukin-1ß.

OBJECTIVE: The aim of this study was to verify the protective effect of hypertonic saline (HS) on brain endothelial cells under hypoxic conditions and the relevant underlying mechanism. METHODS: bEnd.3 cells were treated with oxygen-glucose deprivation (OGD)-induced injury. To measure HS performance, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay, and cell apoptosis was assessed by flow cytometry and Terminal deoxynucleotidyl transferase UTP nick-end labeling staining. RNA-seq was performed to assess the expression profiles and screen the candidate genes that participated in OGD-induced injury and the HS protective effect. Quantitative real-time polymerase chain reaction (qPCR) and western blot analysis were used to confirm the expression of candidate genes, and enzyme-linked immunosorbent assay was used to measure the level of interleukin (IL)-1ß. Overexpression analyses were performed to confirm the functions of the differentially expressed genes. RESULTS: HS with a concentration of 40 mmol/L NaCl had an obvious protective effect on bEnd.3 cells after OGD-induced injury, resulting in increased cell viability and a smaller percentage of apoptotic cells. According to the RNA-seq results, epidermal growth factor receptor (EGFR) was chosen as the differentially expressed gene target in this study. The qPCR and western blot analyses further confirmed that the levels of EGFR/phosphorylated epidermal growth factor receptor and IL-1ß were enhanced after OGD-induced injury, but attenuated after treatment with 40 mmol/L of NaCl HS. Overexpressed EGFR reversed the protective effect of HS that caused low viability and high rates of apoptosis in cells. CONCLUSION: HS can protect endothelial cells against OGD-induced injury, but is affected by the expression of EGFR/p-EGFR and IL-1ß.

Hypertonic saline alleviates experimentally induced cerebral oedema through suppression of vascular endothelial growth factor and its receptor VEGFR2 expression in astrocytes.

BACKGROUND: Cerebral oedema is closely related to the permeability of blood-brain barrier, vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) all of which are important blood-brain barrier (BBB) permeability regulatory factors. Zonula occludens 1 (ZO-1) and claudin-5 are also the key components of BBB. Hypertonic saline is widely used to alleviate cerebral oedema. This study aimed to explore the possible mechanisms underlying hypertonic saline that ameliorates cerebral oedema effectively. METHODS: Middle cerebral artery occlusion (MCAO) model in Sprague-Dawley (SD) rats and of oxygen-glucose deprivation model in primary astrocytes were used in this study. The brain water content (BWC) was used to assess the effect of 10 % HS on cerebral oedema. The assessment of Evans blue (EB) extravasation was performed to evaluate the protective effect of 10 % HS on blood-brain barrier. The quantification of VEGF, VEGFR2, ZO-1 and claudin-5 was used to illustrate the mechanism of 10 % HS ameliorating cerebral oedema. RESULTS: BWC was analysed by wet-to-dry ratios in the ischemic hemisphere of SD rats; it was significantly decreased after 10 % HS treatment (P < 0.05). We also investigated the blood-brain barrier protective effect by 10 % HS which reduced EB extravasation effectively in the peri-ischemic brain tissue. In parallel to the above notably at 24 h following MCAO, mRNA and protein expression of VEGF and VEGFR2 in the peri-ischemic brain tissue was down-regulated after 10 % HS treatment (P < 0.05). Along with this, in vitro studies showed increased VEGF and VEGFR2 mRNA and protein expression in primary astrocytes under hypoxic condition (P < 0.05), but it was suppressed after HS treatment (P < 0.05). In addition, HS inhibited the down-regulation of ZO-1, claudin-5 effectively. CONCLUSIONS: The results suggest that 10 % HS could alleviate cerebral oedema possibly through reducing the ischemia induced BBB permeability as a consequence of inhibiting VEGF-VEGFR2-mediated down-regulation of ZO-1, claudin-5.

Rapamycin inhibits lipopolysaccharide-induced neuroinflammation in vitro and in vivo.

Alzheimer's disease (AD) is the most common type of progressive neurodegenerative disorder, and is responsible for the most common form of dementia in the elderly. Inflammation occurs in the brains of patients with AD, and is critical for disease progression. In the present study, the effects of rapamycin (RAPA) on neuroinflammation lipopolysaccharide (LPS)-induced were investigated. SH­SY5Y human neuroblastoma cells were treated with 20 µg/ml LPS and 0.1, 1 or 10 nmol/l RAPA, and were analyzed at various time points (6, 12 and 24 h). The mRNA expression levels of interleukin (IL) 1ß, IL6 and hypoxia­inducible factor 1α (HIF1α) were determined using reverse transcription­quantitative polymerase chain reaction. The protein expression levels of phosphorylated (p­)S6, p­nuclear factor κB (NFκB), p­inhibitor of NFκB kinase subunit ß (IKKß) and p­tau protein were measured by western blot analysis. p­IKKß, p­NFκB, p­S6 and p­tau were significantly decreased at 6, 12 and 24 h when cells were treated with ≥0.1 nmol/ml RAPA. In addition, female Sprague Dawley rats were intracranially injected with a single dose of 100 µg/kg LPS in the absence or presence of 1 mg/kg RAPA pretreatment. Brain tissues were subjected to immunohistochemical analysis 6­24 h later, which revealed that the expression levels of HIF1α and p­S6 in rat cerebral cortex were increased following LPS injection; however, this increase was abrogated by RAPA treatment. RAPA may therefore be considered a potential therapeutic agent for the early or emergency treatment of neuroinflammation.

The adhesion G protein-coupled receptor GPR56 is a cell-autonomous regulator of oligodendrocyte development.

Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development.

Hypertonic saline alleviates cerebral edema by inhibiting microglia-derived TNF-α and IL-1ß-induced Na-K-Cl Cotransporter up-regulation.

BACKGROUND: Hypertonic saline (HS) has been successfully used clinically for treatment of various forms of cerebral edema. Up-regulated expression of Na-K-Cl Cotransporter 1 (NKCC1) and inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) has been demonstrated to be closely associated with the pathogenesis of cerebral edema resulting from a variety of brain injuries. This study aimed to explore if alleviation of cerebral edema by 10% HS might be effected through down-regulation of inflammatory mediator expression in the microglia, and thus result in decreased NKCC1 expression in astrocytes in the cerebral cortex bordering the ischemic core. METHODS: The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of NKCC1, TNF-α and IL-1ß expression using Western blotting, double immunofluorescence and real time RT-PCR, and the model also was used for evaluation of brain water content (BWC) and infarct size. SB203580 and SP600125, specific inhibitors of the p38 and JNK signaling pathways, were used to treat primary microglia cultures to determine whether the two signaling pathways were required for the inhibition of HS on microglia expressing and secreting TNF-α and IL-1ß using Western blotting, double immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The effect of TNF-α and IL-1ß on NKCC1 expression in primary astrocyte cultures was determined. In addition, the direct inhibitory effect of HS on NKCC1 expression in primary astrocytes was also investigated by Western blotting, double immunofluorescence and real time RT-PCR. RESULTS: BWC and infarct size decreased significantly after 10% HS treatment. TNF-α and IL-1ß immunoexpression in microglia was noticeably decreased. Concomitantly, NKCC1 expression in astrocytes was down-regulated. TNF-α and IL-1ß released from the primary microglia subjected to hypoxic exposure and treatment with 100 mM HS were decreased. NKCC1 expression in primary astrocytes was concurrently and progressively down-regulated with decreasing concentration of exogenous TNF-α and IL-1ß. Additionally, 100 mM HS directly inhibited NKCC1 up-regulation in astrocytes under hypoxic condition. CONCLUSIONS: The results suggest that 10% HS alleviates cerebral edema through inhibition of the NKCC1 Cotransporter, which is mediated by attenuation of TNF-α and IL-1ß stimulation on NKCC1.

Astrocyte-derived proinflammatory cytokines induce hypomyelination in the periventricular white matter in the hypoxic neonatal brain.

Hypoxic exposure in the perinatal period causes periventricular white matter damage (PWMD), a condition associated with myelination abnormalities. Under hypoxic conditions, glial cells were activated and released a large number of inflammatory mediators in the PWM in neonatal brain, which may result in oligodendrocyte (OL) loss and axonal injury. This study aims to determine if astrocytes are activated and generate proinflammatory cytokines that may be coupled with the oligodendroglial loss and hypomyelination observed in hypoxic PWMD. Twenty-four 1-day-old Wistar rats were exposed to hypoxia for 2 h. The rats were then allowed to recover under normoxic conditions for 7 or 28 days before being killed. Another group of 24 rats kept outside the chamber was used as age-matched controls. Upregulated expression of TNF-α and IL-1ß was observed in astrocytes in the PWM of P7 hypoxic rats by double immunofluorescence, western blotting and real time RT-PCR. This was linked to apoptosis and enhanced expression of TNF-R1 and IL-1R1 in APC(+) OLs. PLP expression was decreased significantly in the PWM of P28d hypoxic rats. The proportion of myelinated axons was markedly reduced by electron microscopy (EM) and the average g-ratios were higher in P28d hypoxic rats. Upregulated expression of TNF-α and IL-1ß in primary cultured astrocytes as well as their corresponding receptors in primary culture APC(+) oligodendrocytes were detected under hypoxic conditions. Our results suggest that following a hypoxic insult, astrocytes in the PWM of neonatal rats produce inflammatory cytokines such as TNF-α and IL-1ß, which induce apoptosis of OLs via their corresponding receptors associated with them. This results in hypomyelination in the PWM of hypoxic rats.

Disease-associated mutations prevent GPR56-collagen III interaction.

GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Mutations in GPR56 cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Using the N-terminal fragment of GPR56 (GPR56(N)) as a probe, we have recently demonstrated that collagen III is the ligand of GPR56 in the developing brain. In this report, we discover a new functional domain in GPR56(N), the ligand binding domain. This domain contains four disease-associated mutations and two N-glycosylation sites. Our study reveals that although glycosylation is not required for ligand binding, each of the four disease-associated mutations completely abolish the ligand binding ability of GPR56. Our data indicates that these four single missense mutations cause BFPP mostly by abolishing the ability of GPR56 to bind to its ligand, collagen III, in addition to affecting GPR56 protein surface expression as previously shown.

Amoeboid microglia in the periventricular white matter induce oligodendrocyte damage through expression of proinflammatory cytokines via MAP kinase signaling pathway in hypoxic neonatal rats.

Hypoxic injury in the perinatal period results in periventricular white matter (PWM) lesions with axonal damage and oligodendroglial loss. It also alters macrophage function by perpetuating expression of inflammatory mediators. Relevant to this is the preponderance of amoeboid microglial cells (AMC) characterized as active macrophages in the developing PWM. This study aimed to determine if AMC produce proinflammatory cytokines that may be linked to the oligodendroglial loss observed in hypoxic PWM damage (PWMD). Wistar rats (1 day old) were subjected to hypoxia, following which upregulated expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), TNF receptor 1 (TNF-R(1)) and IL-1 receptor 1 (IL-1R(1)) was observed. This was coupled with apoptosis and expression of TNF-R(1) and IL-1R(1) in oligodendrocytes. Primary cultured microglial cells subjected to hypoxia (3% oxygen, 5% CO(2) and 92% nitrogen) showed enhanced expression of TNF-alpha and IL-1beta. Furthermore, mitogen-activated protein (MAP) kinase signaling pathway was involved in the expression of TNF-alpha and IL-1beta in microglia subjected to hypoxia. Our results suggest that following a hypoxic insult, microglial cells in the neonatal rats produce inflammatory cytokines such as TNF-alpha and IL-1beta via MAP kinase signaling pathway. These cytokines are detrimental to oligodendrocytes resulting in PWM lesion.