Anti-Inflammatory Effect of Ginsenoside Rg1 on LPS-Induced Septic Encephalopathy and Associated Mechanism.

BACKGROUND: Sepsis frequently occurs in patients after infection and is highly associated with death. Septic encephalopathy is characterized by dysfunction of the central nervous system, of which the root cause is a systemic inflammatory response. Sepsis-associated encephalopathy is a severe disease that frequently occurs in children, resulting in high morbidity and mortality. OBJECTIVES: In the present study, we aimed to investigate the neuroprotective mechanism of ginsenoside Rg1 in response to septic encephalopathy. METHODS: Effects of ginsenoside Rg1 on septic encephalopathy were determined by cell viability, cytotoxicity, ROS responses, apoptosis assays, and histological examination of the brain. Inflammatory activities were evaluated by expression levels of IL-1ß, IL-6, IL-10, TNF-α, and MCP-1 using qPCR and ELISA. Activities of signaling pathways in inflammation were estimated by the production of p-Erk1/2/Erk1/2, p-JNK/JNK, p-p38/p38, p-p65/p65, and p-IkBα/IkBα using western blot. RESULTS: LPS simulation resulted in a significant increase in cytotoxicity, ROS responses, and apoptosis and a significant decrease in cell viability in CTX TNA2 cells, as well as brain damage in rats. Moreover, the production of IL-1ß, IL-6, IL-10, TNF-α, and MCP-1 was reported to be significantly stimulated in CTX TNA2 cells and the brain, confirming the establishment of in vitro and in vivo models of septic encephalopathy. The damage and inflammatory responses induced by LPS were significantly decreased by treatment with Rg1. Western blot analyses indicated that Rg1 significantly decreased the production of p-Erk1/2/Erk1/2, p-JNK/JNK, p-p38/p38, p-p65/p65, and p- IkBα/IkBα in LPS-induced CTX TNA2 cells and brain. CONCLUSION: These findings suggested that Rg1 inhibited the activation of NF-κB and MAPK signaling pathways, which activate the production of proinflammatory cytokines and chemokines. The findings of this study suggested that ginsenoside Rg1 is a candidate treatment for septic encephalopathy.

The protective effects of Mirtazapine against lipopolysaccharide (LPS)-induced brain vascular hyperpermeability.

Sepsis is mainly characterized by severe inflammation triggered by infection, and sepsis-associated encephalopathy (SAE) is defined as brain damage caused by sepsis. Disruption of the blood-brain barrier (BBB) triggered by injured brain microvascular endothelial cells (BMECs) and damaged tight junction (TJ) structure is closely associated with the pathogenesis of SAE. The present research proposed to evaluate the potential therapeutic effects of Mirtazapine, a central presynaptic α2 receptor antagonist, on LPS-induced BBB disruption. The mice were administered with normal saline and 10 mg/kg Mirtazapine for 8 consecutive days, and from day 6, the experiment group of mice received LPS for 2 days to induce SAE. We found that the increased BBB permeability, elevated concentrations of inflammatory factors in brain tissues, and downregulated zonula occludens -1 (ZO-1) were observed in LPS-stimulated mice, all of which were reversed by 10 mg/kg Mirtazapine. In the in vitro assay, bEnd.3 brain endothelial cells were treated with 1 µM LPS in the absence or presence of Mirtazapine (25, 50 µM). We found that LPS-treated cells had significantly declined transendothelial electrical resistance (TEER), increased monolayer permeability, elevated production of inflammatory factors, and downregulated ZO-1. However, 25 and 50 µM Mirtazapine ameliorated all these LPS- induced aberrations. Mirtazapine also mitigated the decreased level of NF-E2-related factor 2 (Nrf2) in LPS-challenged endothelial cells. The protective effect of Mirtazapine on endothelial permeability against LPS was significantly abolished by the knockdown of Nrf2. Collectively, we concluded that Mirtazapine exerted protective effects on LPS-induced endothelial cells hyperpermeability by upregulating Nrf2.

Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia.

BACKGROUND: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model. METHODS: Sixteen piglets were randomized: (i) LPS 2 µg/kg bolus; 1 µg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h. RESULTS: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p < 0.01) at 1-3 h and ENO2 (R = -0.69; p = 0.01) at 48 h. CONCLUSIONS: mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE. IMPACT: Early stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. IL1A, CXCL8, IL10, CCL2 and NSE mRNA are promising biomarkers of inflammation-sensitized hypoxia. IL10 mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes.

Impact of ambient temperature on inflammation-induced encephalopathy in endotoxemic mice-role of phosphoinositide 3-kinase gamma.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is an early and frequent event of infection-induced systemic inflammatory response syndrome. Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and inflammation-related microglial activity. In homeotherms, variations in ambient temperature (Ta) outside the thermoneutral zone lead to thermoregulatory responses, mainly driven by a gradually increasing sympathetic activity, and may affect disease severity. We hypothesized that thermoregulatory response to hypothermia (reduced Ta) aggravates SAE in PI3Kγ-dependent manner. METHODS: Experiments were performed in wild-type, PI3Kγ knockout, and PI3Kγ kinase-dead mice, which were kept at neutral (30 ± 0.5 °C) or moderately lowered (26 ± 0.5 °C) Ta. Mice were exposed to lipopolysaccharide (LPS, 10 µg/g, from Escherichia coli serotype 055:B5, single intraperitoneal injection)-evoked systemic inflammatory response (SIR) and monitored 24 h for thermoregulatory response and blood-brain barrier integrity. Primary microglial cells and brain tissue derived from treated mice were analyzed for inflammatory responses and related cell functions. Comparisons between groups were made with one-way or two-way analysis of variance, as appropriate. Post hoc comparisons were made with the Holm-Sidak test or t tests with Bonferroni's correction for adjustments of multiple comparisons. Data not following normal distribution was tested with Kruskal-Wallis test followed by Dunn's multiple comparisons test. RESULTS: We show that a moderate reduction of ambient temperature triggers enhanced hypothermia of mice undergoing LPS-induced systemic inflammation by aggravated SAE. PI3Kγ deficiency enhances blood-brain barrier injury and upregulation of matrix metalloproteinases (MMPs) as well as an impaired microglial phagocytic activity. CONCLUSIONS: Thermoregulatory adaptation in response to ambient temperatures below the thermoneutral range exacerbates LPS-induced blood-brain barrier injury and neuroinflammation. PI3Kγ serves a protective role in suppressing release of MMPs, maintaining microglial motility and reinforcing phagocytosis leading to improved brain tissue integrity. Thus, preclinical research targeting severe brain inflammation responses is seriously biased when basic physiological prerequisites of mammal species such as preferred ambient temperature are ignored.

ProBDNF promotes sepsis-associated encephalopathy in mice by dampening the immune activity of meningeal CD4+ T cells.

BACKGROUND: Sepsis-associated encephalopathy (SAE) increases the mortality of septic patients, but its mechanism remains unclear. The present study aimed to investigate the roles of T lymphocytes, proBDNF, and their interaction in the pathogenesis of SAE. METHODS: Fear conditioning tests were conducted for cognitive assessment in the lipopolysaccharide (LPS, 5 mg kg-1)-induced septic mice. Meninges and peripheral blood were harvested for flow cytometry or qPCR. FTY720 and monoclonal anti-proBDNF antibody (McAb-proB) were used to investigate the effect of lymphocyte depletion and blocking proBDNF on the impaired cognitive functions in the septic mice. RESULTS: In the septic mice, cognitive function was impaired, the percentage of CD4+ T cells were decreased in the meninges (P = 0.0021) and circulation (P = 0.0222), and pro-inflammatory cytokines were upregulated, but the anti-inflammatory cytokines interleukin (IL)-4 (P < 0.0001) and IL-13 (P = 0.0350) were downregulated in the meninges. Lymphocyte depletion by intragastrically treated FTY720 (1 mg kg-1) for 1 week ameliorated LPS-induced learning deficit. In addition, proBDNF was increased in the meningeal (P = 0.0042) and peripheral (P = 0.0090) CD4+ T cells. Intraperitoneal injection of McAb-proB (100 µg) before LPS treatment significantly alleviated cognitive dysfunction, inhibited the downregulation of meningeal (P = 0.0264) and peripheral (P = 0.0080) CD4+ T cells, and normalized the gene expression of cytokines in the meninges. However, intra-cerebroventricular McAb-proB injection (1 µg) did not have such effect. Finally, exogenous proBDNF downregulated the percentage of CD4+ T cells in cultured splenocytes from septic mice (P = 0.0021). CONCLUSION: Upregulated proBDNF in immune system promoted the pathogenesis of SAE through downregulating the circulating CD4+ T cells, limiting its infiltration into the meninges and perturbing the meningeal pro-/anti-inflammatory homeostasis.

Incidence, risk factors, and outcomes for sepsis-associated delirium in patients with mechanical ventilation: A sub-analysis of a multicenter randomized controlled trial.

PURPOSE: This study aimed to investigate incidence, risk factors, and outcomes for sepsis-associated delirium (SAD) in mechanically ventilated patients. MATERIALS AND METHODS: We performed a retrospective post-hoc analysis of the DExmedetomidine for Sepsis in Intensive care unit Randomized Evaluation (DESIRE) trial. Outcomes included 28-day mortality, ventilator-free days, length of ICU stay, self-extubation, and re-intubation. Multivariable analysis was performed to identify variables independently associated with SAD. RESULTS: We retrospectively divided the patients into two groups: delirium group (n = 89) and non-delirium group (n = 98). There were no significant differences between the groups in 28-day mortality, self-extubation, and re-intubation. The number of ventilator-free days was significantly less in the delirium vs. non-delirium group (17 vs. 22 days, p = .006), and the length of ICU stay was significantly longer in the delirium group (10 vs. 5 days, p = .04). Multivariable analyses revealed that emergency surgery, more doses of midazolam, and fentanyl were independent predictors for SAD. CONCLUSIONS: SAD was associated with a less number of ventilator-free days and longer length of ICU stay. Emergency surgery, more doses of midazolam, and fentanyl may be independent risk factors for SAD in mechanically ventilated patients with sepsis.

Widespread time-dependent changes in tissue cytokine concentrations in brain regions during the acute phase of endotoxemia in mice.

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction induced by the systemic response to infection in septic patients. In the present study, we modeled SAE by administering lipopolysaccharide (LPS) intraperitoneally to mice at a concentration of 3.0 mg/kg. We investigated regional preferences for cytokine-mediated brain reactions to endotoxemia and at what time point brain inflammation begins, as well as what cytokines are involved in acute brain reactions. Brains were divided into seven parts: cortex (CTX), olfactory system (Olf), hippocampus (Hip), striatum (Str), diencephalon (Die), brain stem (BS), and cerebellum (CBL). In each brain region, we determined the tissue concentrations of 11 cytokines: CCL2, CCL3, CCL11, CXCL1, CXCL2, CXCL9, CXCL10, G-CSF, IL-1ß, IL-6, and TNF-α, in mice injected with LPS or saline, at 1, 4, and 24 h after injection using multiplex cytokine assays. Every brain region responded with the production of multiple cytokines to LPS-induced systemic inflammation during the acute phase (4-24 h) after LPS injection. IL-6, CCL2, CCL3, CXCL1, CXCL2, CXCL9, and TNF-α were "early cytokines" that increased only at 4 h but not at 24 h after LPS injection in most brain regions. CCL11, CXCL10, and G-CSF were "late cytokines" that were elevated up to 24 h after LPS injection in selected brain regions. The regions Olf, Hip, and Die were the most responsive to endotoxemia; these regions produced ten cytokines and continued to produce three "late cytokines" up to 24 h after LPS injection. Str was the least responsive to endotoxemia. The widespread nature of brain cytokine production explains the characteristics of SAE. Further studies on the roles of CCL11, CXCL10, and G-CSF may be especially important in terms of potential prevention of SAE between 4 and 24 h after the onset of sepsis.

Disruption of Striatal-Enriched Protein Tyrosine Phosphatase Signaling Might Contribute to Memory Impairment in a Mouse Model of Sepsis-Associated Encephalopathy.

Sepsis-associated encephalopathy (SAE) is a potentially irreversible acute cognitive dysfunction with unclear mechanism. Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase which normally opposes synaptic strengthening by regulating key signaling molecules involved in synaptic plasticity and neuronal function. Thus, we hypothesized that abnormal STEP signaling pathway was involved in sepsis-induced cognitive impairment evoked by lipopolysaccharides (LPS) injection. The levels of STEP, phosphorylation of GluN2B (pGluN2B), the kinases extracellular signal-regulated kinase 1/2 (pERK), cAMP-response element binding protein (CREB), synaptophysin, brain derived neurotrophic factor (BDNF), and post-synaptic density protein 95 (PSD95) in the hippocampus, prefrontal cortex, and striatum were determined at the indicated time points. In the present study, we found that STEP levels were significantly increased in the hippocampus, prefrontal cortex, and striatum following LPS injection, which might resulted from the disruption of the ubiquitin-proteasome system. Notably, a STEP inhibitor TC-2153 treatment alleviated sepsis-induced memory impairment by increasing phosphorylation of GluN2B and ERK1/2, CREB/BDNF, and PSD95. In summary, our results support the key role of STEP in sepsis-induced memory impairment in a mouse model of SAE, whereas inhibition of STEP may provide a novel therapeutic approach for this disorder and possible other neurodegenerative diseases.

Altered functional connectivity within the default mode network in two animal models with opposing episodic memories.

Memory enhancement and memory decline are two opposing cognitive performances commonly observed in clinical practice, yet the neural mechanisms underlying these two different phenomena remain poorly understood. Accumulating evidence has demonstrated that the default-mode network (DMN) is implicated in diverse cognitive, social, and affective processes. In the present study, we used the retrosplenial cortex as a seed region to study the functional connectivity within the DMN in two animal models with opposing episodic memories, of which memory enhancement was induced by footshocks to mimic posttraumatic stress disorder (PTSD) and memory decline was induced by lipopolysaccharide (LPS) challenge to mimic sepsis-associated encephalopathy (SAE). Our results showed that LPS challenge and footshocks induced opposing episodic memories. With regard to the imaging data, there were significant differences in the functional connectivity between the retrosplenial cortex and the medial prefrontal cortex (mPFC), insular lobe, left piriform cortex, left sensory cortex, and right visual cortex among the three groups. Post-hoc comparisons showed the LPS group had a significantly increased functional connectivity between the retrosplenial cortex and mPFC as compared with the control group. Compared with the LPS group, the PTSD group displayed significantly decreased functional connectivity between the retrosplenial cortex and the right visual cortex, retrosplenial cortex, insular lobe, left piriform cortex, and left sensory cortex. In summary, our study suggests that there is a significant difference in the functional connectivity within the DMN between SAE and PTSD rats.

Evaluation of Brain Nuclear Medicine Imaging Tracers in a Murine Model of Sepsis-Associated Encephalopathy.

PURPOSE: The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as possible methods to study the early effects of systemic inflammation on the living brain in a mouse model of sepsis-associated encephalopathy (SAE). The lipopolysaccharide (LPS)-induced murine systemic inflammation model was selected as a model of SAE. PROCEDURES: C57BL/6 mice were used. A multimodal imaging protocol was carried out on each animal 4 h following the intravenous administration of LPS using the following tracers: [99mTc][2,2-dimethyl-3-[(3E)-3-oxidoiminobutan-2-yl]azanidylpropyl]-[(3E)-3-hydroxyiminobutan-2-yl]azanide ([99mTc]HMPAO) and ethyl-7-[125I]iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([125I]iomazenil) to measure brain perfusion and neuronal damage, respectively; 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) to measure cerebral glucose uptake. We assessed microglia activity on another group of mice using 2-[6-chloro-2-(4-[125I]iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-methyl-acetamide ([125I]CLINME). Radiotracer uptakes were measured in different brain regions and correlated. Microglia activity was also assessed using immunohistochemistry. Brain glutathione levels were measured to investigate oxidative stress. RESULTS: Significantly reduced perfusion values and significantly enhanced [18F]FDG and [125I]CLINME uptake was measured in the LPS-treated group. Following perfusion compensation, enhanced [125I]iomazenil uptake was measured in the LPS-treated group's hippocampus and cerebellum. In this group, both [18F]FDG and [125I]iomazenil uptake showed highly negative correlation to perfusion measured with ([99mTc]HMPAO uptake in all brain regions. No significant differences were detected in brain glutathione levels between the groups. The CD45 and P2Y12 double-labeling immunohistochemistry showed widespread microglia activation in the LPS-treated group. CONCLUSIONS: Our results suggest that [125I]CLINME and [99mTc]HMPAO SPECT can be used to detect microglia activation and brain hypoperfusion, respectively, in the early phase (4 h post injection) of systemic inflammation. We suspect that the enhancement of [18F]FDG and [125I]iomazenil uptake in the LPS-treated group does not necessarily reflect neural hypermetabolism and the lack of neuronal damage. They are most likely caused by processes emerging during neuroinflammation, e.g., microglia activation and/or immune cell infiltration.

Individual differences in the brain are associated with resilience versus susceptibility to lipopolysaccharide-induced memory impairment.

Sepsis impairs learning and memory function, yet marked interindividual variability exists in the degree to which sepsis compromises learning and memory function. Thus, testing resilience versus susceptibility to systemic inflammation induced-memory impairment and the underlying mechanism is needed. In the present study, we firstly used lipopolysaccharide (LPS) to induce memory impairment, and then evaluated cognitive function on days 4-7 after the first LPS challenge. Subjects' scores on both behavioral measures were subjected to a hierarchical cluster analysis, identifying two clusters that differed notably on the Y-maze and fear conditioning tests. This analysis divided these subjects into two groups, one cluster (13 of 34 subjects) displayed impaired working and associative memory, named "Susceptive". The remaining cluster (21 of 34 subjects) showed normal memory, named "Resilient". We have also included another group receiving normal saline to serve as the control group. The three groups underwent a battery of biochemical detections. In addition, we investigated whether the individual differences would disappear between the "Resilient" and "Susceptive" groups by using microglia inhibitor minocycline. We showed that as compared with the "Resilient" or control group, the "Susceptive" group was accompanied by increased tumor necrosis factor-alpha, interleukin-1beta (IL-1ß), IL-6, and biomarkers of microglia activation ionized calcium binding adaptor molecule-1 and cluster of differentiation 68. Notably, after decreasing the activation of microglia, the differences in cognitive function between the "Resilient" and "Susceptive" groups disappeared. Collectively, our study suggests that individual differences in the brain are associated with resilience versus susceptibility to LPS-induced memory impairment.

Huperzine A protects sepsis associated encephalopathy by promoting the deficient cholinergic nervous function.

Neuroinflammatory deregulation in the brain plays a crucial role in the pathogenesis of sepsis associated encephalopathy (SAE). Given the mounting evidence of anti-inflammatory and neuroprotective effects of the cholinergic nervous system, it is surprising that there is little information about its changes in the brain during sepsis. To elucidate the role of the cholinergic nervous system in SAE, hippocampal choline acetyltransferase, muscarinic acetylcholine receptor-1, acetylcholinesterase and acetylcholine were evaluated in LPS-induced sepsis rats. Expression of pro-inflammatory cytokines, neuronal apoptosis, and animal cognitive performance were also assessed. Furthermore, therapeutic effects of the acetylcholinesterase inhibitor Huperzine A (HupA) on the hippocampal cholinergic nervous function and neuroinflammation were evaluated. A deficiency of the cholinergic nervous function was revealed in SAE, accompanied with over-expressed pro-inflammatory cytokines, increase in neuronal apoptosis and brain cognitive impairment. HupA remarkably promoted the deficient cholinergic nervous function and attenuated the abnormal neuroinflammation in SAE, paralleled with the recovery of brain function. We suggest that the deficiency of the cholinergic nervous function and the abnormal neuroinflammation are synergistically implicated in the pathogenesis of SAE. Thus, HupA is a potential therapeutic candidate for SAE, as it improves the deficient cholinergic nervous function and exerts anti-inflammatory action.