Bacterial DNA metabolism analysis by metagenomic next-generation sequencing (mNGS) after treatment of bloodstream infection.

BACKGROUND: With the advent of metagenomic next-generation sequencing (mNGS), the time of DNA metabolism can be explored after bacteria be killed. In this study, we applied mNGS in investigation of the clearance profile of circulating bacteria DNA. METHODS: All of the rabbits were injected with the inactivated Escherichia coli. Using mNGS, we analyzed serial samples of plasma collected from rabbits to detect clearance profile of circulating E. coli DNA. RESULTS: In this study, we found that the of E. coli DNA could still be detected 6 h after injecting killed bacteria. The clearance half-lives associated with the 2 phases are 0.37 and 1.81 h. We also explored there is no correlation between the disease severity with the E. coli DNA reads in circulation. CONCLUSIONS: After the bacteria were completely killed, their DNA could still be detected in the blood circulation. The metabolism of bacterial DNA in the circulation had two phases: fast and slow phases, and there were no correlations between the level of bacteria reads with the severity of patients' disease after the bacteria have been completely killed.

Systemic immune-inflammation index predicted the clinical outcome in patients with type-B aortic dissection undergoing thoracic endovascular repair.

BACKGROUND: The systemic immune-inflammation index (SII) has been reported to have prognostic ability in various cardiovascular diseases; however, it has not been studied in type-B aortic dissection (TBAD). We aimed to explore the relation of SII with short-term and long-term outcomes in TBAD patients undergoing thoracic endovascular repair (TEVAR). METHODS: We performed a retrospective analysis of a prospectively maintained database from 2010 to 2017. The patients were divided into two groups (high SII and low SII) as per the optimal cut-off value determined using the receiver operating characteristic curve. Multivariate logistic and Cox regression analyses were performed to analyse the relationship between the SII and the short-term and long-term outcomes. RESULTS: A total of 805 TBAD patients who underwent TEVAR were enrolled. Twenty-six (3.2%) patients died during hospitalisation. At the end of a median follow-up duration of 48.80 mon, 70 (9.8%) patients had died. The patients were divided into the high-SII group [n = 333 (41.4%%)] and the low-SII group [n = 472 (58.6%)] as per the optimal cut-off value of 1,062. Multivariable logistic analyses showed that a high-SII score was independently associated with major adverse cardiovascular events (MACEs) in-hospital (odd ratio [OR], 1.67; 95% confidence interval [CI], 1.13-2.47; p = .01). In addition, multivariable Cox analyses showed that a high-SII score could be an independent indicator for follow-up adverse events (hazard ratio [HR], 1.70; 95% CI, 1.14-2.56, p = .01). CONCLUSIONS: Systemic immune-inflammation index is associated with both in-hospital and long-term outcomes in patients with TBAD undergoing TEVAR. Therefore, SII may serve as valuable tool for risk stratification before intervention.

Bradykinin/bradykinin 1 receptor promotes brain microvascular endothelial cell permeability and proinflammatory cytokine release by downregulating Wnt3a.

Stroke is a life-threatening disease with limited therapeutic options. Damage to the blood-brain barrier (BBB) is the key pathological feature of ischemic stroke. This study explored the role of the bradykinin (BK)/bradykinin 1 receptor (B1R) and its mechanism of action in the BBB. Human brain microvascular endothelial cells (BMECs) were used to test for cellular responses to BK by using the Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining, enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, cellular permeability assays, and western blotting to evaluate cell viability, cytokine production, and reactive oxygen species (ROS) levels in vitro. A BBB induced by middle cerebral artery occlusion was used to evaluate BBB injuries, and the role played by BK/B1R in ischemic/reperfusion (I/R) was explored in a rat model. Results showed that BK reduced the viability of BMECs and increased the levels of proinflammatory cytokines (interleukin 6 [IL-6], IL-18, and monocyte chemoattractant protein-1) and ROS. Additionally, cellular permeability was increased by BK treatment, and the expression of tight junction proteins (claudin-5 and occludin) was decreased. Interestingly, Wnt3a expression was inhibited by BK and exogenous Wnt3a restored the effects of BK on BMECs. In an in vivo I/R rat model, knockdown of B1R significantly decreased infarct volume and inflammation in I/R rats. Our results suggest that BK might be a key inducer of BBB injury and B1R knockdown might provide a beneficial effect by upregulating Wnt3a.

Arterial catheterization and in-hospital mortality in sepsis: a propensity score-matched study.

BACKGROUND: Despite the extensive use of arterial catheterization (AC), clinical effectiveness of AC to alter the outcomes among patients with sepsis and septic shock has not been evaluated. The purpose of this study is to examine the association between the use of AC and in-hospital mortality in septic patients. METHODS: Adult patients with sepsis from Medical Information Mart for Intensive Care database were screened to conduct this retrospective observational study. Propensity score matching (PSM) was employed to estimate the relationship between arterial catheterization (AC) and in-hospital mortality. Multivariable logistic regression and inverse probability of treatment weighing (IPTW) were used to validate our findings. RESULTS: A total of 14,509 septic patients without shock and 4,078 septic shock patients were identified. 3,489 pairs in sepsis patients without shock and 589 pairs in septic shock patients were yielded respectively after PSM. For patients in the sepsis without shock group, AC placement was associated with increased in-hospital mortality (OR, 1.34; 95% CI, 1.17-1.54; p < 0.001). In the septic shock group, there was no significant difference in hospital mortality between AC group and non-AC group. The results of logistic regression and propensity score IPTW model support our findings. CONCLUSIONS: In hemodynamically stable septic patients, AC is independently associated with higher in-hospital mortality, while in patients with septic shock, AC was not associated with improvements in hospital mortality.

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.

Prognostic dynamic nomogram integrated with metabolic acidosis for in-hospital mortality and organ malperfusion in acute type B aortic dissection patients undergoing thoracic endovascular aortic repair.

BACKGROUND: Organ malperfusion is a lethal complication in acute type B aortic dissection (ATBAD). The aim of present study is to develop a nomogram integrated with metabolic acidosis to predict in-hospital mortality and organ malperfusion in patients with ATBAD undergoing thoracic endovascular aortic repair (TEVAR). METHODS: The nomogram was derived from a retrospectively study of 286 ATBAD patients who underwent TEVAR from 2010 to 2017 at a single medical center. Model performance was evaluated from discrimination and calibration capacities, as well as clinical effectiveness. The results were validated using a prospective study on 77 patients from 2018 to 2019 at the same center. RESULTS: In the multivariate analysis of the derivation cohort, the independent predictors of in-hospital mortality and organ malperfusion identified were base excess, maximum aortic diameter ≥ 5.5 cm, renal dysfunction, D-dimer level ≥ 5.44 µg/mL and albumin amount ≤ 30 g/L. The penalized model was internally validated by bootstrapping and showed excellent discriminatory (bias-corrected c-statistic, 0.85) and calibration capacities (Hosmer-Lemeshow P value, 0.471; Brier Score, 0.072; Calibration intercept, - 0.02; Slope, 0.98). After being applied to the external validation cohort, the model yielded a c-statistic of 0.86 and Brier Score of 0.097. The model had high negative predictive values (0.93-0.94) and moderate positive predictive values (0.60-0.71) for in-hospital mortality and organ malperfusion in both cohorts. CONCLUSIONS: A predictive nomogram combined with base excess has been established that can be used to identify high risk ATBAD patients of developing in-hospital mortality or organ malperfusion when undergoing TEVAR.

Hypercapnia Exacerbates the Blood-Brain Barrier Disruption Via Promoting HIF-1a Nuclear Translocation in the Astrocytes of the Hippocampus: Implication in Further Cognitive Impairment in Hypoxemic Adult Rats.

Hypercapnia in combination with hypoxemia is usually present in severe respiratory disease in the intensive care unit (ICU) and can lead to more severe cognitive dysfunction. Increasing evidence has indicated that the compromised blood-brain barrier (BBB) in the hippocampus in hypoxemia conditions can result in cognitive dysfunction. However, the role and underlying mechanism of hypercapnia in the BBB disruption remains poorly known. A rat model of hypercapnia was first established in this study by intubation and mechanical ventilation with a small-animal ventilator. After this, the cognitive function of the experimental rats was assessed by the Morris water maze test. The BBB permeability was evaluated by the Evans Blue (EB) test and brain water content (BWC). Western blot analysis was carried out to detect the protein expressions of total and nuclear hypoxia-inducible factor-1α (HIF-1α), matrixmetalloproteinase-9 (MMP-9) and Aquaporins-4 (AQP-4) in the hippocampus tissue. Double immunofluorescence further verified the protein expression of different biomarkers was localized in the astrocytes of the hippocampus. Hypercapnia alone did not disrupt the BBB, but it could further enhance the BBB permeability in hypoxemia. Concomitantly, up-regulation of nuclear HIF-1α, AQP-4, MMP-9 protein expression along with increased degradation of the occludin and claudin-5 proteins was found in the hypercapnia rat model, while the total HIF-1α remained unchanged. Interestingly, these changes were independent of the acidosis induced by hypercapnia. Of note, after premedication of 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α nuclear translocation), the disrupted BBB could be restored resulting in improvement of the cognitive impairment. Meanwhile, accumulation of nuclear HIF-1α, protein expression of AQP-4 and MMP-9 and protein degradation of the occludin and claudin-5 were decreased. Thus, our study demonstrated that hypercapnia can further disrupt the BBB through promoting HIF-1α nuclear translocation and up-regulation of AQP-4 and MMP-9 in hypoxemia. It is therefore suggested that the cascade of hypercapnia-induced nuclear HIF-1α protein translocation in hypoxia-activated astrocytes may be a potential target for ameliorating cognitive impairment.

Increased percentage of PD-L1+ natural killer cells predicts poor prognosis in sepsis patients: a prospective observational cohort study.

BACKGROUND: Natural killer (NK) cells play a major role in immune tolerance after sepsis, and the programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) system mediates evasion of host immunity. The correlation between PD-L1 levels in NK cells and the prognosis of patients with sepsis, however, has not been elucidated. Thus, it was hypothesized that PD-L1 in NK cells could be a novel biomarker of the mortality for sepsis patients. METHODS: A prospective, observational, cohort study in a general intensive care unit had earlier enrolled patients according to the sepsis-3 criteria, and peripheral blood samples were collected within 24 h post-recruitment. The expression of four co-signaling molecules (PD-1, CD28, PD-L1, and CD86) in NK cells was assayed, and the sequential organ failure assessment (SOFA) scores were recorded on day 1. Patients were followed up until 28 days. Multivariate regression analysis assessed the independent risk factors for 28-day mortality. The association between biomarkers and 28-day mortality was assessed by Cox regression survival analysis. The accuracy of biomarkers for mortality was determined by the area under the receiver operating characteristic (ROC) curve (AUC) analysis. RESULTS: A total of 269 patients were recruited, and 114 patients were finally included for final analysis. Of these, 30 (26.3%) patients died during 28 days. The percentage of PD-L1+ NK cells (OR 1.022; 95% CI 1.002-1.043) and SOFA scores (OR 1.247; 95% CI 1.092-1.424) were independent risk factors for 28-day mortality. The AUC of the percentage of PD-L1+ NK cells, SOFA scores, and their combination model were 0.655 (0.559-0.742), 0.727 (0.635-0.807) and 0.808 (0.723-0.876), respectively. The combination model was the indicator with the best AUC to predict mortality in 28 days (all p < 0.05). Patients with the percentage of PD-L1+ NK cells above the cutoff point 5.58% (hazard ratio (HR) 10.128 (1.372-74.772), p = 0.001), and the combination model prediction possibility above 0.1241 (HR 13.730 (3.241-58.158), p < 0.001) were the indexes that had greater discriminative capacity to predict 28 days mortality. CONCLUSIONS: The percentage of PD-L1+ NK cells at admission serves as a novel prognostic biomarker for predicting mortality and contributes to improve the predictive capacity of SOFA score in patients with sepsis.

Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 in cerebral edema treatment.

BACKGROUND: Hypertonic saline (HS) has been used clinically for treatment of cerebral edema for decades. Previously we have demonstrated that HS alleviates cerebral edema via regulating water/ion channel protein and attenuating neuroinflammation. However, whether HS treatment triggers microglia polarization and its regulatory mechanism during this process is unclear. METHODS AND RESULTS: The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of neuroinflammation and microglia functions. Treatment of 10% HS not only significantly reduced infarct size and ipsilateral ischemic hemispheric brain water content (BWC) via attenuating ischemia-induction of TNF-α, IL-1ß, microglia M1 markers (iNOS, CD86) and miR-200b, but also increased neurotrophic factors such as IL-10 and IL-4, microglia M2 markers (Arg1, CD206) and Krüppel-like factor 4 (KLF4). Similar changes were confirmed in primary microglial cells subjected to hypoxia with/without HS in vitro. Importantly, overexpression of miR-200b was able to induce microglia M1 polarization via directly targeting KLF4. Restoring KLF4 expression abolished this effect. On the contrary, miR-200b inhibitor or KLF4 overexpression led to microglia M2 polarization. Mechanistically, KLF4 directly binds to promoter region of Agr1, thus inducing its transcription. Similar to treatment of HS, experimental overexpression of KLF4 in vivo exerted significant beneficial effects on ischemia-induced cerebral edema. However, knockdown of KLF4 abrogated the benefits of HS. CONCLUSIONS: Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 during its treatment of cerebral edema. This study may provide new insights of HS-related therapy for cerebral edema.

Hypercapnia induces IL-1ß overproduction via activation of NLRP3 inflammasome: implication in cognitive impairment in hypoxemic adult rats.

BACKGROUND: Cognitive impairment is one of common complications of acute respiratory distress syndrome (ARDS). Increasing evidence suggests that interleukin-1 beta (IL-1ß) plays a role in inducing neuronal apoptosis in cognitive dysfunction. The lung protective ventilatory strategies, which serve to reduce pulmonary morbidity for ARDS patients, almost always lead to hypercapnia. Some studies have reported that hypercapnia contributes to the risk of cognitive impairment and IL-1ß secretion outside the central nervous system (CNS). However, the underlying mechanism of hypercapnia aggravating cognitive impairment under hypoxia has remained uncertain. This study was aimed to explore whether hypercapnia would partake in increasing IL-1ß secretion via activating the NLRP3 (NLR family, pyrin domain-containing 3) inflammasome in the hypoxic CNS and in aggravating cognitive impairment. METHODS: The Sprague-Dawley (SD) rats that underwent hypercapnia/hypoxemia were used for assessment of NLRP3, caspase-1, IL-1ß, Bcl-2, Bax, and caspase-3 expression by Western blotting or double immunofluorescence, and the model was also used for Morris water maze test. In addition, Z-YVAD-FMK, a caspase-1 inhibitor, was used to treat BV-2 microglia to determine whether activation of NLRP3 inflammasome was required for the enhancing effect of hypercapnia on expressing IL-1ß by Western blotting or double immunofluorescence. The interaction effects were analyzed by factorial ANOVA. Simple effects analyses were performed when an interaction was observed. RESULTS: There were interaction effects on cognitive impairment, apoptosis of hippocampal neurons, activation of NLRP3 inflammasome, and upregulation of IL-1ß between hypercapnia treatment and hypoxia treatment. Hypercapnia + hypoxia treatment caused more serious damage to the learning and memory of rats than those subjected to hypoxia treatment alone. Expression levels of Bcl-2 were reduced, while that of Bax and caspase-3 were increased by hypercapnia in hypoxic hippocampus. Hypercapnia markedly increased the expression of NLRP3, caspase-1, and IL-1ß in hypoxia-activated microglia both in vivo and in vitro. Pharmacological inhibition of NLRP3 inflammasome activation and release of IL-1ß might ameliorate apoptosis of neurons. CONCLUSIONS: The present results suggest that hypercapnia-induced IL-1ß overproduction via activating the NLRP3 inflammasome by hypoxia-activated microglia may augment neuroinflammation, increase neuronal cell death, and contribute to the pathogenesis of cognitive impairments.

Evaluation of a combination "lymphocyte apoptosis model" to predict survival of sepsis patients in an intensive care unit.

BACKGROUND: A major challenge in sepsis intervention is unclear risk stratification. We postulated that a panel of biomarkers of lymphocyte apoptosis and immune function, termed the "lymphocyte apoptosis model," would be an effective tool for predicting 28-day survival for sepsis patients. METHODS: A total of 52 consecutive sepsis patients were enrolled. Peripheral blood samples were collected on day 1 of admission for quantification of biomarkers of lymphocyte apoptosis and immune function, including lymphocyte count, lymphocyte apoptotic percentage, expression on monocyte HLA-DR, CD4+/CD8+ T cell ratio, T helper type 1 to type 2 ratio (Th1/Th2), cytochrome c levels, and various proinflammatory cytokine levels. Sepsis severity was classified using Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores. Survival was assessed at 28 days. RESULTS: Compared with survivors, non-survivors had significantly higher lymphocyte apoptotic percentages and plasma cytochrome c levels and significantly lower lymphocyte counts, Th1/Th2 ratios, and HLA-DR expression on day 1 of admission. Multivariate analysis identified cytochrome c levels (odds ratio [OR]1.829, p = 0.025), lymphocyte apoptotic percentage (OR 1.103, p = 0.028), lymphocyte count (OR 0.150, p = 0.047), and HLA-DR expression (OR 0.923, p = 0.021) as independent predictors of 28-day mortality. A logistic regression equation incorporating the independent risk factors predicted 28-day mortality with greater accuracy than did the APACHE II score or single components biomarkers. CONCLUSIONS: The "lymphocyte apoptosis model" may be useful for risk stratification and predicting prognosis of sepsis patients.

Microglia-derived IL-1ß contributes to axon development disorders and synaptic deficit through p38-MAPK signal pathway in septic neonatal rats.

BACKGROUND: Axon development plays a pivotal role in the formation of synapse, nodes of Ranvier, and myelin sheath. Interleukin-1ß (IL-1ß) produced by microglia may cause myelination disturbances through suppression of oligodendrocyte progenitor cell maturation in the septic neonatal rats. Here, we explored if a microglia-derived IL-1ß would disturb axon development in the corpus callosum (CC) following lipopolysaccharide (LPS) administration, and if so, whether it is associated with disorder of synapse formation in the cerebral cortex and node of Ranvier. METHODS: Sprague-Dawley rats (1-day old) in the septic model group were intraperitoneally administrated with lipopolysaccharide (1 mg/kg) and then sacrificed for detection of IL-1ß, interleukin-1 receptor (IL-1R1), neurofilament-68, neurofilament-160, and neurofilament-200, proteolipid, synaptophysin, and postsynaptic density 95 (PSD95) expression by western blotting and immunofluorescence. Electron microscopy was conducted to observe alterations of axonal myelin sheath and synapses in the cortex, and proteolipid expression was assessed using in situ hybridization. The effect of IL-1ß on neurofilament and synaptophysin expression in primary neuron cultures was determined by western blotting and immunofluorescence. P38-MAPK signaling pathway was investigated to determine whether it was involved in the inhibition of IL-1ß on neurofilament and synaptophysin expression. RESULTS: In 1-day old septic rats, IL-1ß expression was increased in microglia coupled with upregulated expression of IL-1R1 on the axons. The expression of neurofilament-68, neurofilament-160, and neurofilament-200 (NFL, NFM, NFH) and proteolipid (PLP) was markedly reduced in the CC at 7, 14, and 28 days after LPS administration. Simultaneously, cortical synapses and mature oligodendrocytes were significantly reduced. By electron microscopy, some axons showed smaller diameter and thinner myelin sheath with damaged ultrastructure of node of Ranvier compared with the control rats. In the cerebral cortex of LPS-injected rats, some axo-dendritic synapses appeared abnormal looking as manifested by the presence of swollen and clumping of synaptic vesicles near the presynaptic membrane. In primary cultured neurons incubated with IL-1ß, expression of NFL, NFM, and synaptophysin was significantly downregulated. Furthermore, p38-MAPK signaling pathway was implicated in disorder of axon development and synaptic deficit caused by IL-1ß treatment. CONCLUSIONS: The present results suggest that microglia-derived IL-1ß might suppress axon development through activation of p38-MAPK signaling pathway that would contribute to formation disorder of cortical synapses and node of Ranvier following LPS exposure.

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.

IL-1ß induces hypomyelination in the periventricular white matter through inhibition of oligodendrocyte progenitor cell maturation via FYN/MEK/ERK signaling pathway in septic neonatal rats.

Neuroinflammation elicited by microglia plays a key role in periventricular white matter (PWM) damage (PWMD) induced by infectious exposure. This study aimed to determine if microglia-derived interleukin-1ß (IL-1ß) would induce hypomyelination through suppression of maturation of oligodendrocyte progenitor cells (OPCs) in the developing PWM. Sprague-Dawley rats (1-day old) were injected with lipopolysaccharide (LPS) (1 mg/kg) intraperitoneally, following which upregulated expression of IL-1ß and IL-1 receptor 1 (IL-1R1 ) was observed. This was coupled with enhanced apoptosis and suppressed proliferation of OPCs in the PWM. The number of PDGFR-α and NG2-positive OPCs was significantly decreased in the PWM at 24 h and 3 days after injection of LPS, whereas it was increased at 14 days and 28 days. The protein expression of Olig1, Olig2, and Nkx2.2 was significantly reduced, and mRNA expression of Tcf4 and Axin2 was upregulated in the developing PWM after LPS injection. The expression of myelin basic protein (MBP) and 2',3'-cyclic-nucleotide 3"-phosphodiesterase (CNPase) was downregulated in the PWM at 14 days and 28 days after LPS injection; this was linked to reduction of the proportion of myelinated axons and thinner myelin sheath as revealed by electron microscopy. Primary cultured OPCs treated with IL-1ß showed the failure of maturation and proliferation. Furthermore, FYN/MEK/ERK signaling pathway was involved in suppression of maturation of primary OPCs induced by IL-1ß administration. Our results suggest that following LPS injection, microglia are activated and produce IL-1ß in the PWM in the neonatal rats. Excess IL-1ß inhibits the maturation of OPCs via suppression of FYN/MEK/ERK phosphorylation thereby leading to axonal hypomyelination.

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.

Validation of the use of B-type natriuretic peptide point-of-care test platform in preliminary recognition of cardioembolic stroke patients in the ED.

AIM: The aim of the study is to validate of the use of plasma B-type natriuretic peptide (BNP) point-of-care test platform in preliminary recognition of cardioembolic stroke patients in the emergency department (ED). METHODS: In our ED, emergency physicians prospectively assessed consecutive adult patients with acute phase of ischemic stroke and measured plasma BNP by point-of-care test platform on admission. The included patients with plasma BNP concentration more than 66.50 pg/mL were presumed to be classified as the cardioembolism (CE) subtype and were then followed up. Stroke neurologists evaluated patients' functional outcome at hospital discharge and also made discharge diagnosis and stroke etiologic subtypes according to Trial of ORG 10172 in Acute Stroke Treatment criteria: large artery atherosclerosis, CE, small artery occlusion, stroke of other determined etiology, and stroke of other undetermined etiology. RESULTS: In this study, 172 of 262 acute ischemic stroke patients met the study criteria (mean age, 71.18 ± 11.65 years; 53.49% female). Of the 172 patients, 38.95% were diagnosed with large artery atherosclerosis at discharge; 26.16%, with CE; 24.42%, with small artery occlusion; and 10.47%, with stroke of other determined etiology or stroke of other undetermined etiology. Age, previous cardiac disease, atrial fibrillation, length of hospital stays, Scandinavian Stroke Scale score on admission less than or equal to 25, and modified Rankin Scale greater than or equal to 3 or death at discharge were all significantly higher in the CE patients compared to other subtypes (P < .01). The mean BNP concentration was significantly higher in the CE group than in other 3 subtypes (P < .01). The plasma BNP level greater than 66.50 pg/mL had good corresponding diagnostic performance in preliminary recognition of cardioembolic stroke patients (sensitivity, 75.56%; specificity, 87.40%). CONCLUSIONS: In this study, we found that the plasma BNP level greater than 66.50 pg/mL as a reference index had good corresponding diagnostic performance in preliminary recognition of cardioembolic stroke patients. However, the single BNP biomarker panel cannot be used to confidently identify CE subtype as a diagnosis and must be taken in context with clinical assessment and judgment before making management decisions.

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.

Hyperglycemia induces microglial pyroptosis by increasing oxygen extraction rate: Implication in neurological impairment during ischemic stroke.

Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia promotes microglial pyroptosis by increasing the oxygen extraction rate in an acute ischemic stroke model. C57BL/6 mice that underwent middle cerebral artery occlusion were used for assessment of blood glucose level and neurological function. The cerebral oxygen extraction ratio (CERO2), oxygen consumption rate (OCR) and partial pressure of brain tissue oxygen (PbtO2) were measured. To investigate the significance of the NOD­like receptor protein 3 (NLRP3) inflammasome, NLRP3­/­ mice were used, and the expression levels of NLRP3, caspase­1, full­length gasdermin D (GSDMD­FL), GSDMD­N domain (GSDMD­N), IL­1ß and IL­18 were evaluated. In addition, Z­YVAD­FMK, a caspase­1 inhibitor, was used to treat microglia to determine whether activation of the NLRP3 inflammasome was required for the enhancing effect of hyperglycemia on pyroptosis. It was revealed that hyperglycemia accelerated cerebral injury in the acute ischemic stroke model, as evidenced by decreased latency to fall and the percentage of foot fault. Hyperglycemia aggravated hypoxia by increasing the oxygen extraction rate, as evidenced by increased CERO2 and OCR, and decreased PbtO2 in response to high glucose treatment. Furthermore, hyperglycemia­induced microglial pyroptosis was confirmed by detection of increased levels of caspase­1, GSDMD­N, IL­1ß and IL­18 and a decreased level of GSDMD­FL. However, the knockout of NLRP3 attenuated these effects. Pharmacological inhibition of caspase­1 also reduced the expression levels of GSDMD­N, IL­1ß and IL­18 in microglial cells. These results suggested that hyperglycemia stimulated NLRP3 inflammasome activation by increasing the oxygen extraction rate, thus leading to the aggravation of pyroptosis following ischemic stroke.

PD-1 mediates microglia polarization via the MAPK signaling pathway to protect blood-brain barrier function during cerebral ischemia/reperfusion.

BACKGROUND: Cerebral ischemia is characterized by its rapid onset and high rates of recurrence, morbidity, and mortality, with blood-brain barrier (BBB) permeability playing a vital role in brain injury. Therefore, it is important to understand the molecular mechanism which regulates the BBB during cerebral ischemia. MATERIALS AND METHODS: An in vitro model of oxygen-glucose deprivation (OGD) and an in vivo model of cerebral ischemia/reperfusion (I/R) were constructed. PD-1 overexpression vectors and vectors containing si-RNA were transfected and injected into in vitro and in vivo models. Western blotting, real-time quantitative PCR (qPCR), immunofluorescence (IF) analysis, and immunohistochemical staining were employed to evaluate the expression levels of programmed cell death-1 (PD-1), microglia M1 and M2 biomarkers, and tight junction proteins. Flow cytometry and ELISA were used to measure the levels of pro-inflammatory cytokines. The BBB permeability of brain tissues was evaluated by Evans blue dye (EBD) extravasation and transendothelial electrical resistance (TEER). Brain water content was measured to assess the extent of inflammatory exudation. The infarct volume and neurological severity score (NSS) were used to assess the severity of brain injury. Brain cell apoptosis was assessed by the TUNEL assay and hematoxylin-eosin (H&E) staining. RESULTS: PD-1 helped to convert the microglia M1 phenotype to the M2 phenotype and to reduce BBB permeability both in vitro and in vivo. Overexpression of PD-1 promoted a shift of the M1 phenotype to the M2 phenotype and reduced BBB permeability via the ERK and p38 MAPK signaling pathways. PD-1 reduced inflammatory exudation, BBB permeability, cell apoptosis, and brain injury in vivo. CONCLUSION: Our present study verified that PD-1 exerts an anti-inflammatory effect by converting the microglia M1 phenotype to the M2 phenotype, reducing BBB permeability, and thereby relieves brain injury caused by cerebral ischemia. PD-1 is potential therapeutic target for brain injury caused by cerebral ischemia.