Optimal Targets of the First 24-h Partial Pressure of Carbon Dioxide in Patients with Cerebral Injury: Data from the MIMIC-III and IV Database.

BACKGROUND: It is generally believed that hypercapnia and hypocapnia will cause secondary injury to patients with craniocerebral diseases, but a small number of studies have shown that they may have potential benefits. We assessed the impact of partial pressure of arterial carbon dioxide (PaCO2) on in-hospital mortality of patients with craniocerebral diseases. The hypothesis of this research was that there is a nonlinear correlation between PaCO2 and in-hospital mortality in patients with craniocerebral diseases and that mortality rate is the lowest when PaCO2 is in a normal range. METHODS: We identified patients with craniocerebral diseases from Medical Information Mart for Intensive Care third and fourth edition databases. Cox regression analysis and restricted cubic splines were used to examine the association between PaCO2 and in-hospital mortality. RESULTS: Nine thousand six hundred and sixty patients were identified. A U-shaped association was found between the first 24-h PaCO2 and in-hospital mortality in all participants. The nadir for in-hospital mortality risk was estimated to be at 39.5 mm Hg (p for nonlinearity < 0.001). In the subsequent subgroup analysis, similar results were found in patients with traumatic brain injury, metabolic or toxic encephalopathy, subarachnoid hemorrhage, cerebral infarction, and other encephalopathies. Besides, the mortality risk reached a nadir at PaCO2 in the range of 35-45 mm Hg. The restricted cubic splines showed a U-shaped association between the first 24-h PaCO2 and in-hospital mortality in patients with other intracerebral hemorrhage and cerebral tumor. Nonetheless, nonlinearity tests were not statistically significant. In addition, Cox regression analysis showed that PaCO2 ranging 35-45 mm Hg had the lowest death risk in most patients. For patients with hypoxic-ischemic encephalopathy and intracranial infections, the first 24-h PaCO2 and in-hospital mortality did not seem to be correlated. CONCLUSIONS: Both hypercapnia and hypocapnia are harmful to most patients with craniocerebral diseases. Keeping the first 24-h PaCO2 in the normal range (35-45 mm Hg) is associated with lower death risk.

Fisetin ameliorates cognitive impairment by activating mitophagy and suppressing neuroinflammation in rats with sepsis-associated encephalopathy.

BACKGROUND: Fisetin, the effective ingredient of the traditional Chinese medicine named Cotinus coggygria, is recommended to be active therapeutic in many disorders. However, its role in sepsis-associated encephalopathy (SAE) remains unclarified. METHODS: Cecal ligation and puncture (CLP) operation was performed to establish a rat model of SAE. Rats were grouped according to the surgery operation and fisetin administration. Cognitive impairment was assessed by Morris water maze test. Disruption of blood-brain barrier (BBB) integrity was detected by Evan's blue staining. The mitophagy, reactive oxygen species (ROS) generation, NLRP3 inflammasome activation, and pro-inflammatory cytokines levels were measured through western blot and double immunofluorescence labeling. A transmission electron microscope was applied for the observation of mitochondrial autophagosomes. RESULTS: Rats in the CLP group presented increased expression of IL-1R1, pNF-κB, TNF-α, and iNOS in microglial cells, indicating severe inflammation in the central nervous system (CNS). Nevertheless, there was no increase in BBB permeability. Meanwhile, NLRP3 inflammasome was activated in cerebral microvascular endothelial cells (CMECs), presented with an elevation of caspase-1 expression and IL-1ß secretion into CNS. In addition, we found fisetin significantly improved cognitive dysfunction in rats with SAE. Neuroprotective effects of fisetin might be associated with inhibition of neuroinflammation, represented with decreased expression of IL-1R1, pNF-κB, TNF-α, and iNOS in microglia. Furthermore, fisetin induced mitophagy, scavenged ROS, blocked NLRP3 inflammasome activation of CMECs, as evidenced by decreased expression of caspase-1 and reduced release of IL-1ß into CNS. CONCLUSION: Collectively, fisetin-blocked NLRP3 inflammasome activation via promoting mitophagy in CMECs may suppress the secretion of IL-1ß into CNS, reduce neuroinflammation, and contribute to the amelioration of cognitive impairment.

Risk stratification of ulcer-like projection in uncomplicated acute type B aortic intramural haematoma.

OBJECTIVES: This study aimed to investigate the morphological evolution and risk stratification of ulcer-like projection (ULP) in patients with uncomplicated acute type B aortic intramural haematoma. METHODS: A retrospective study was conducted on patients with uncomplicated acute type B intramural haematoma admitted in our institution from January 2015 to June 2020. The primary end points were adverse aortic events (AAE), including aortic rupture, aortic dissection, aortic aneurysm and ULP enlargement. RESULTS: The study cohort comprised 140 patients, including 62 (44%) and 78 (56%) patients with and without initial ULP, respectively. AAE occurred in 13 patients (9%) in the early term and 42 patients (33%) in the mid-term. Compared with patients without ULP, patients with initial ULP had no significant difference in early outcomes but a higher mid-term AAE rate [8% vs 11%, odds ratio (OR) 1.5, P = 0.47; 17% vs 55%, OR 6.0, P < 0.001]. Significantly higher AAE rate was observed in patients with high-risk ULP (depth ≥5.0 mm and located in the proximal aortic segments) than those with only low-risk ULP (depth <5.0 mm and/or located in the distal aortic segments) (87% vs 51%, OR 6.2, P = 0.014). In the multivariable analysis, high-risk ULP was an independent predictor of AAE (hazard ratio 2.8, P = 0.009). CONCLUSIONS: High-risk ULP is a rapidly evolving entity and a marker of AAE despite optimal medical therapy. Therefore, close follow-up and prompt intervention are recommended for patients with high-risk ULP.

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.

Peri-operative risk factors for in-hospital mortality in acute type A aortic dissection.

BACKGROUND: Acute type A aortic dissection (TAAD) is cardiovascular emergency and requires surgical interventions. In-hospital mortality rate of surgical-treated TAAD patients remains high. We aim to examine the prognostic implications of peri-operative parameters to identify high-risk patient for in-hospital mortality. METHODS: A total of 264 surgically treated TAAD patients were included in this study. The association between in-hospital mortality and peri-operative parameters were examined. RESULTS: Thirty patients (11.36%) died during hospitalization. Patients with higher Apache II score had a significantly higher rate of in-hospital mortality when compared with patients scored ≤20 in unadjusted model [Score 21-25: HR =12.9 (1.7-100.8), P=0.0148; Score >25: HR =94.5 (12.6-707.6), P<0.0001]. Patients with Sbp >120 mmHg, Cr >200 mmol/L (both at admission and after surgery), BUN >8.2 mmol/L (both at admission and after surgery), AST >80 µ/L, aortic cross-clamping time >120 min and cardiopulmonary bypass time (CPBT) >230 min were also significantly related to higher rate of in-hospital mortality in univariate analysis. In multivariable analysis, APACHE II score [Score 21-25: HR =9.5 (1.2-74.4), P=0.032; Score >25: HR =51.0 (6.7-387.7), P=0.0001], AST >80 µmol/L [HR =2.3 (1.1-4.8), P=0.0251], aortic cross-clamping time >120 min (HR =2.9 (1.1-7.7), P=0.0315) remained significant in predicting TAAD in-hospital mortality. CONCLUSIONS: APACHE II score could be a useful tool to predict TAAD in-hospital mortality. AST >80 µ/L and aortic cross-clamping time >120 min were also independent predictors.

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.

[ß1 receptor blocker decreases the myocardial inflammation in the sepsis adult rats through inhibition of TLR4/NF-ΚB signaling pathway].

OBJECTIVE: To explore whether ß1 receptor blocker could decrease the myocardial inflammation through the Toll-like receptor 4/nuclear factor-ΚB (TLR4/NF-ΚB) signaling pathway in the sepsis adult rats. METHODS: Sixty male Wistar rats (250-300 g) aged 3 months old were allocated to four groups by random number table (n = 15): sham operation group (S group), sepsis model group (CLP group), ß1 receptor blocker esmolol intervention group (ES group), and inhibitor of the TLR4 E5564 intervention group (E5564 group). The rat sepsis model was established by cecal ligation and puncture (CLP); S group of rats underwent only an incision. Rats in S group, CLP group and E5564 group were subcutaneous injected with 0.9% sodium chloride (NaCl) 2.0 mL/kg. Besides, the rats in ES group were injected with esmolol (15 mg×kg-1×h-1) by micro pump through the caudal vein. The rats in E5564 group were injected with E5564 (0.3 mg×kg-1×h-1) by micro pump through the caudal vein 1 hour before the CLP surgery. Samples were collected 6 hours after the modelling in each group. The average arterial pressure (MAP) and cardiac output index (CI) were monitored by PU electrical conduction ECG monitor. The levels of serum cardiac troponin I (cTnI), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). The expressions of TLR4, NF-ΚB p65, IL-1ß, TNF-α in myocardial tissue was detected by Western Blot. RESULTS: There was no significant difference in MAP in each group. Compared with the S group, the CI in the CLP group was significantly decreased, the levels of serum cTnI, IL-1ß, TNF-α were significantly increased, the protein expressions of myocardial tissue TLR4, NF-ΚB p65, IL-1ß and TNF-α were significantly increased. Compared with the CLP group, the CI in the ES group and E5564 group were significantly increased (mL×s-1×m-2: 58.6±4.3, 58.9±4.4 vs. 41.2±3.9, both P < 0.01), the levels of serum cTnI, IL-1ß and TNF-α were significantly decreased [cTnI (µg/L): 1 113.81±26.64, 1 115.74±25.90 vs. 1 975.96±42.74; IL-1ß (ng/L): 39.6±4.3, 38.9±4.4 vs. 61.2±3.9; TNF-α (ng/L): 43.1±2.8, 48.7±2.6 vs. 81.3±4.4, all P < 0.01], the protein expressions of myocardial tissue NF-ΚB p65, IL-1ß, TNF-α were significantly decreased (NF-ΚB p65/ß-actin: 0.31±0.03, 0.43±0.04 vs. 0.85±0.08; IL-1ß/ß-actin: 0.28±0.05, 0.32±0.03 vs. 0.71±0.06; TNF-α/ß-actin: 0.18±0.04, 0.28±0.03 vs. 0.78±0.07, all P < 0.01), but there was no significant difference in protein expression of TLR4 (TLR4/ß-actin: 0.89±0.07, 0.87±0.09 vs. 0.95±0.09, both P > 0.05). There was no significant difference in CI, the levels of serum cTnI, IL-1ß, TNF-α, and the protein expressions of myocardial tissue TLR4, NF-ΚB p65, IL-1ß, TNF-α between ES group and E5564 group (all P > 0.05). CONCLUSIONS: ß1 receptor blocker esmolol may inhibit myocardial inflammatory response in sepsis adult rats through TLR4/NF-ΚB signaling pathway, thereby alleviating sepsis-induced myocardial injury.

NF-κB mediates early blood-brain barrier disruption in a rat model of traumatic shock.

BACKGROUND: Blood-brain barrier (BBB) disruption is associated with a large number of central nervous system and systemic disorders. The aim of the present study was to investigate the dynamic change of BBB changes during traumatic shock and resuscitation as well as the mechanisms involved. METHODS: The experiments were performed on male Sprague-Dawley rats anesthetized with pentobarbital sodium. To produce traumatic shock, the rats were subjected to bilateral femoral traumatic fracture and blood withdrawal from the femoral artery to decrease mean arterial pressure (MAP) to 35 mm Hg. Hypovolemic status (at a MAP of 35 to 40 mm Hg) was sustained for 1 hour followed by fluid resuscitation with shed blood and 20 mL/kg of lactated Ringer's solution. RESULTS: The rats were sacrificed at 1 hour, 2 hours, or 6 hours after fluid resuscitation. Blood-brain barrier permeability studies showed that traumatic shock significantly increased brain water contents and sodium fluorescein leakage, which was aggravated by fluid resuscitation. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses revealed that Na-K-Cl cotransporter-1 and vascular endothelial growth factor (VEGF) expression were upregulated in cortical brain tissue of traumatic shock rats, and this change was accompanied by downregulation of occludin and claudin-5. Traumatic shock also significantly increased the protein levels of NF-κB-p65 subunit. Of note, administration of NF-κB inhibitor PDTC effectively attenuated augmentation of the above changes. CONCLUSION: Our results suggest that traumatic shock is associated with early BBB disruption, and inhibition of NF-κB may be an effective therapeutic strategy in protecting the BBB under traumatic shock conditions.

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.

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.

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.

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.

[Cholinergic anti-inflammatory pathway involves in the neuroprotective effect of huperzine A on sepsis-associated encephalopathy].

Objective: To explore whether the cholinergic anti-inflammatory pathway is involved in the neuroprotective effect of acetylcholinesterase inhibitor huperzine A (HupA) on sepsis-associated encephalopathy (SAE) by observing the effect of HupA on the expressions of choline acetyltransferase (CHAT) and cholinergic muscarinic receptor M1 (CHRM1) of sepsis rats. Methods: Fifty-four male Wistar rats, 8 weeks old, were divided into three experimental groups according to random number table:control group,sepsis group, and HupA group, with 18 rats in each group. The rat model of sepsis was reproduced by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS,1 mL),and the rats in control group were given the same volume of normal saline. The rats in HupA group were intraperitoneally administered with HupA 0.04 mg/kg (1 mL) at 30 minutes before model reproduction, while the rats in control group and sepsis group were treated with the same volume of saline instead. At 3,12,and 24 hours after model reproduction,6 rats in each group were sacrificed after clinical manifestation observation, and cerebral cortex tissue was collected. Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1 ß) in cerebral cortex were determined with enzyme linked immunosorbent assay (ELISA),along with the measurement of neuronal apoptosis by caspase-3 and neuronal nuclear antigen (NeuN) immune double standard staining. The mRNA expressions and positive expressions of ChAT and CHRM1 were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunofluorescence methods. Results: Clinical manifestations of sepsis rats were present at 3 hours and reached a peak at 12 hours, including lethargy,vertical hair and lazy to move. Over-expression of pro-inflammatory cytokines TNF-α and IL-1ß was found in sepsis group, and apoptotic neurons marked by two fluorescences were significantly increased in sepsis rats ,in comparison with deficient ChAT and CHRM1 proteins marked by red fluorescence, and low-expressed ChAT and CHRM1 mRNA as well. The differences between sepsis group and control group were statistically significant [12-hour TNF-α (ng/L):84.97±31.84 vs.40.31 ± 10.37,12-hour IL-1 ß (ng/L):1 095.98± 127.09 vs.622.62± 117.25,12-hour ChAT mRNA (2-△△Ct):1.34 (0.67,1.86) vs.1.92 (1.12,2.87),12-hour CHRM1 mRNA (2-△△Ct):0.65±0.12 vs.1.16±0.42,all P ＜ 0.05].The septic symptoms were relieved after HupA administration,as well as the reduction of pro-inflammatory cytokines and the neuronal apoptosis, which might attribute to the increased expressions of ChAT and CHRM1.The differences between HupA group and sepsis group were statistically significant [12-hour TNF-α (ng/L):48.38 ± 12.62 vs.84.97 ± 31.84,12-hour IL-1 ß (ng/L):718.13 ± 163.33 vs.1 095.98 ± 127.09,12-hour ChAT mRNA (2-△△Ct):18.04 (17.22,19.23) vs.1.34 (0.67,1.86),12-hour CHRM1 mRNA (2-△ △Ct):1.46 ± 0.69 vs 0.65 ± 0.12,all P ＜ 0.05].The clinical manifestations and neuroinflammation mainly recovered at 24 hours. Conclusions: The cortical cholinergic neurons dysfunction and the abnormal inflammatory response are involved in the onset of SAE process. HupA plays a protective role in SAE through recovering the function of cholinergic neurons and cholinergic anti-inflammatory pathway.

[Significance of the ratio of plasma vascular endothelial growth factor level to platelet count in the prognosis of patients with sepsis].

OBJECTIVE: To investigate the clinical value of the ratio of plasma vascular endothelial growth factor level to platelet count (VEGF/PLT) in predicting 28-day prognosis in patients with sepsis. METHODS: A prospective cohort study was conducted. From September 2009 to March 2013, 164 sepsis patients in Intensive Care Unit (ICU) of Guangdong General Hospital were included for study. Patients with age younger than 18 years old, the illness already reaching final stage of chronic diseases, suffering from two or more organs dysfunction within 3 days, acute pancreatitis without infection, or less than 28 days of expected survival time were excluded. Finally, 135 patients were included in the further analysis. Peripheral blood samples were collected at admission. Routine blood tests were done, and then VEGF levels in plasma were measured by enzyme linked immunosorbent assay (ELISA). Acute physiology and chronic health evaluation II (APACHEII) scores were recorded every day for 7 days. Patients' prognosis was assessed during the following 28 days. The patients were divided into 28-day survival group and non-survival group. Comparison between two groups was done by single factor analysis. Spearman rank correlation was used to analyze the correlation between VEGF levels and PLT. Mutivariate logistic regression analysis was performed to identify the independent risk factor for 28-day prognosis. Receiver operating characteristic curve (ROC curve) was plotted, and the effect of related indexes on predicting 28-day survival was evaluated by area under ROC curve (AUC). RESULTS: There were no significant differences in VEGF (471.73±198.34 ng/L vs. 383.49±266.54 ng/L, t=-1.918, P=0.057), PLT (220.40±127.60×10(9)/L vs. 246.42±100.72×10(9)/L, t=1.275, P=0.204), leucocyte counts (12.48±4.62×10(9)/L vs. 13.70±5.97×10(9)/L, t=1.063, P=0.292), mean arterial pressure (86.50±12.04 mmHg vs. 91.03±13.10 mmHg, t=1.557, P=0.123) and blood lactic acid (1.79±1.30 mmol/L vs. 1.50±0.60 mmol/L, t=-1.768, P=0.079) at admission between the non-survival group (n=42) and survival group (n=93). VEGF/PLT (2.59±1.44 vs. 1.73±1.13, t=-3.756, P=0.000) as well as APACHEII scores (15.50±4.50 vs. 13.28±4.61, t=-2.022, P=0.045) of the non-survival group were significantly higher than those of survival group, and oxygenation index (PaO2/FiO2) of the non-survival group was significantly lower than that of survival group (32.38±11.12 kPa vs. 37.04±10.97 kPa, t=2.278, P=0.024). Correlation analysis showed that the concentration of VEGF was positively correlated with PLT (r=0.271, P=0.001). It was shown by multivariate logistic regression analysis that only VEGF/PLT was the independent risk factor in predicting 28-day prognosis in patients with sepsis [odds ratio (OR) was 1.591, 95% confidence interval (95%CI) 1.164-2.175, P=0.004]. AUC of VEGF/PLT was 0.704±0.047 (P=0.000, 95%CI: 0.611-0.797) for predicting 28-day survival. The optimal cut-off point was 1.32, and the sensitivity and specificity were 81.0% and 48.4 %,respectively. CONCLUSIONS: VEGF/PLT can be used as one of the indicators to predict 28-day survival in patients with sepsis.

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.

[Effect of hydroxyethyl starch on intracranial pressure and plasma colloid osmotic pressure in rats with cerebral ischemia/reperfusion injury].

OBJECTIVE: To investigate the regulatory effect of hydroxyethyl starch on colloidal osmotic pressure (COP), and its effect on intracranial pressure (ICP) in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: Twenty four male Sprague Dawley (SD) rats were randomly divided into sham operation group, model group and the hydroxyethyl starch group, each n =8. Cerebral I/R model was reproduced by middle cerebral artery occlusion (MCAO), followed by reperfusion after ischemia for 2 hours. Rats in hydroxyethyl starch group received hydroxyethyl starch 130/0.4206 ml × kg(-1)× d(-1) ia tail vein at the beginning of reperfusion. ICP and COP were evaluated at 0, 2, 6, 12, 18, 24 hours after the surgery. The rats were sacrificed by decapitation. The water content of the right hemisphere was measured at 24 hours after the surgery, and the ratio of apoptosis of neurons was observed by immunohistochemical method. RESULTS: Two hours after surgery the ICP (mm Hg, 1 mm Hg=0.133 kPa) of model group and hydroxyethyl starch group was significantly increased compared with sham operation group (11.50 ± 1.43, 12.48 ± 0.75 vs. 7.95 ± 0.92, both P <0.05). With prolongation of time, the ICP gradually increased and reached the peak at 24 hours (22.76 ± 0.72, 23.32 ± 0.98 vs. 8.15 ± 1.09, both P <0.05). But there was no significant difference in ICP in the hydroxyethyl starch group compared with that of the model group at all time points. The COP (mm Hg) of hydroxyethyl starch group was significantly higher than the model group and sham operation group at each time point, and peaked at 6 hours after surgery (13.49 ± 0.50 vs. 12.04 ± 0.47, 12.00 ± 0.39, both P <0.01). There was no significant difference in COP between the model group and the sham operation group at all time points. The brain water content, neuronal apoptosis of hydroxyethyl starch group and model group was significantly higher than sham operation group [brain water content: (80.16 ± 0.44)%, (80.59 ± 0.67)% vs. (78.72 ± 0.52)%; neuronal apoptosis:(44.27 ± 7.86)%,(42.82 ± 7.82)%vs. (3.26 ± 0.00)%, P <0.05 or P <0.01], but there was no significant difference between the hydroxyethyl starch group and model group (both P >0.05). CONCLUSION: Intravenous injection of hydroxyethyl starch 130/0.4 can increase the plasma COP, but it can not significantly reduce ICP and brain water content, and it also can not improve the neuronal apoptosis.