GSDMD/Drp1 signaling pathway mediates hippocampal synaptic damage and neural oscillation abnormalities in a mouse model of sepsis-associated encephalopathy.

BACKGROUND: Gasdermin D (GSDMD)-mediated pyroptotic cell death is implicated in the pathogenesis of cognitive deficits in sepsis-associated encephalopathy (SAE), yet the underlying mechanisms remain largely unclear. Dynamin-related protein 1 (Drp1) facilitates mitochondrial fission and ensures quality control to maintain cellular homeostasis during infection. This study aimed to investigate the potential role of the GSDMD/Drp1 signaling pathway in cognitive impairments in a mouse model of SAE. METHODS: C57BL/6 male mice were subjected to cecal ligation and puncture (CLP) to establish an animal model of SAE. In the interventional study, mice were treated with the GSDMD inhibitor necrosulfonamide (NSA) or the Drp1 inhibitor mitochondrial division inhibitor-1 (Mdivi-1). Surviving mice underwent behavioral tests, and hippocampal tissues were harvested for histological analysis and biochemical assays at corresponding time points. Haematoxylin-eosin staining and TUNEL assays were used to evaluate neuronal damage. Golgi staining was used to detect synaptic dendritic spine density. Additionally, transmission electron microscopy was performed to assess mitochondrial and synaptic morphology in the hippocampus. Local field potential recordings were conducted to detect network oscillations in the hippocampus. RESULTS: CLP induced the activation of GSDMD, an upregulation of Drp1, leading to associated mitochondrial impairment, neuroinflammation, as well as neuronal and synaptic damage. Consequently, these effects resulted in a reduction in neural oscillations in the hippocampus and significant learning and memory deficits in the mice. Notably, treatment with NSA or Mdivi-1 effectively prevented these GSDMD-mediated abnormalities. CONCLUSIONS: Our data indicate that the GSDMD/Drp1 signaling pathway is involved in cognitive deficits in a mouse model of SAE. Inhibiting GSDMD or Drp1 emerges as a potential therapeutic strategy to alleviate the observed synaptic damages and network oscillations abnormalities in the hippocampus of SAE mice.

Cuscuta chinensis Lam. Flavonoids (CCLF) alleviate the symptoms of sepsis-associated encephalopathy via PI3K/Nrf2 pathway.

Sepsis-associated encephalopathy (SAE) frequently encounters patients who are in intensive care units and ∼70% of patients with severe systemic infection. However, due to the unclear pathological mechanisms of SAE, the desease-modifying drug is still lack. Here, we aimed to explore whether the flavonoid components extracted from CCL (CCLF) seeds possess protective effects on SAE animals, and systematically evaluate the transcriptomic alteration (in the hippocampus) after CCLF treatment on SAE animals employing RNA sequencing. We observed that CCLF improved the brain's learning and memory abilities and the structural integrity of BBB using cecal ligation and puncture (CLP)-induced SAE animal models, evaluated by behavioral test and tissue examination of animals respectively. RNA sequencing results showed that CCLF treatment reverses SAE-induced transcriptomic alteration in the hippocampus. Moreover, CCLF also dramatically relieved inflammatory (such as TNF-α, IL-2, and IL-6) and oxidative (MDA and SOD activity) stresses, and inhibited SAE-induced neuron apoptosis in brain tissues. More importantly, CCLF restored the PI3K/AKT signaling pathway and then induced the Nrf2 nuclear translocation to drive HO-1 expression both in vitro and in vivo. LY294002, an inhibitor of PI3K, obviously blocked CCLF's functions on anti-apoptosis, anti-inflammation, and anti-oxidation in vivo, demonstrating that CCLF achieves its bioactivities in a PI3K/AKT signaling dependent manner. Altogether, CCLF exhibits remarkable neuro-protective function and may be a promising candidate for further clinical trials for SAE treatment.

Maresin1 improves hippocampal neuroinflammation and cognitive function in septic rats by activating the SLC7A11 / GPX4 ferroptosis signaling pathway.

Sepsis-associated encephalopathy (SAE) is a prevalent complication of sepsis, with hippocampal neuroinflammation playing a crucial role in SAE-induced cognitive impairment. Maresin1 (MaR1), a bioactive docosahexaenoic acid (DHA) metabolite, demonstrates comprehensive anti-inflammatory and neuroprotective attributes. Yet, its protective efficacy against SAE-induced cognitive decline remains unexplored. In this investigation, we implemented a rat SAE model via cecal ligation and puncture (CLP), while lipopolysaccharide (LPS) stimulation of HT22 cells simulated an in vitro SAE model; both models were pre-treated with MaR1. We evaluated rat learning and memory using a water maze, assessed hippocampal neuron damage via Nissl and FJC staining, and observed mitochondrial alterations through TEM. In vivo and in vitro assays gauged levels of Fe2+, MDA, GSH, and SOD. Additionally, Iba1 expression in the hippocampus was examined via immunofluorescence, while SLC7A11 and GPX4 protein expression levels were determined using western blot. Our findings indicated CLP-induced learning and memory impairment in rats, along with heightened ROS, Fe2+, and MDA levels in hippocampal neurons, diminished GSH and SOD levels, and down-regulated ferroptosis-related proteins (GPX4 and SLC7A11). Remarkably, MaR1 treatment attenuated these adverse effects. In LPS-stimulated HT22 cells, MaR1 lowered lipid ROS and bolstered mitochondrial membrane potential. Nonetheless, the ferroptosis inducer Erastin reversed MaR1's protective effects. Transwell experiments further showed MaR1's potential to inhibit microglia activation triggered by ferroptosis in HT22 cells. Consequently, MaR1 may mitigate hippocampal neuroinflammation via activating the SLC7A11/GPX4 ferroptosis signaling pathway, thus ameliorating SAE-related cognitive impairment.

Meta-analysis of evaluating neuron specific enolase as a serum biomarker for sepsis-associated encephalopathy.

INTRODUCTION: Brain dysfunction in sepsis is known as Sepsis-associated encephalopathy (SAE), which often results in severe cognitive and neurological sequelae and increases the risk of death. Neuron specific enolase (NSE) may serve as an important neurocritical biomarker for detection and longitudinal monitoring in SAE patients. Our Meta-analysis aimed to explore the diagnostic and prognostic value of serum NSE in SAE patients. Currently, no systematic Review and Meta-analysis have been assessed that NSE as a biomarker of SAE. METHODS: The study protocol was registered in the PROSPERO database (CRD42023398736) and adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We conducted a systematic review and Meta-analysis to evaluate the serum NSE's diagnostic accuracy for SAE and prognostic strength for probability of death of septic patients. We systematic searched electronic bibliographic databases from PubMed, MEDLINE, Web of Science, Embase, Cochrane databases, CNKI, CQVIP, and WFSD. QUADAS-2 assessment tool was used to evaluate quality and risk of bias of the selected studies. Subgroup analyses, funnel plots, sensitivity analyses were also carried out. Review Manager version 5.4 and Stata16.0. was used for statistical analysis. RESULTS: This Meta-analysis included 22 studies with 1361 serum samples from SAE patients and 1580 serum samples from no-encephalopathy septic (NE) patients. The Meta-analysis showed that individuals with SAE had higher serum NSE level than NE controls (SMD 1.93 (95 % CI 1.51-2.35), P < 0.00001). In addition, there are 948 serum samples from survival septic patients and 446 serum samples from non-survival septic patients, septic patients with survival outcomes had lower serum NSE levels than those with death outcomes (SMD -1.87 (95 % CI -2.43 to -1.32), P < 0.00001). CONCLUSION: Our Meta-analysis reveals a significant association between elevated NSE concentrations and the increased likelihood of concomitant SAE and mortality during septic patients. This comprehensive analysis will equip ICU physicians with up-to-date insights to accurately identify patients at risk of SAE and implement appropriate intervention strategies to mitigate morbidity and improve neurological outcomes. However, it is important to note that the presence of substantial heterogeneity among studies poses challenges in determining the most effective discrimination cutoff values and optimal sampling collection time.

Cholinesterase activities and sepsis-associated encephalopathy in viral versus nonviral sepsis.

PURPOSE: There is evidence that cholinergic imbalance secondary to neuroinflammation plays a role in the pathophysiology of sepsis-associated encephalopathy (SAE). Blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities have been proposed as surrogate parameters for the cholinergic function of the central nervous system. Viral sepsis is associated with systemic inflammation and BChE has been reported to be of prognostic value in a small cohort of COVID-19 patients. Nevertheless, the prognostic value of AChE in patients with viral sepsis remains unclear. METHODS: We investigated the role of AChE and BChE activities as prognostic biomarkers of SAE and mortality in patients with viral vs nonviral sepsis enrolled in two prospective cohort studies. We quantified the AChE and BChE activities in whole blood of patients at two time points in the acute phase of viral sepsis (N = 108) and compared them with the activities in patients with nonviral sepsis (N = 117) and healthy volunteers (N = 81). Patients were observed until discharge from the intensive care unit (ICU). RESULTS: Three days after sepsis onset, the median [interquartile range] levels of AChE and BChE were reduced in both patients with viral sepsis (AChE, 5,105 [4,010-6,250] U·L-1; BChE, 1,943 [1,393-2,468] U·L-1) and nonviral sepsis (AChE, 4,424 [3,630-5,055] U·L-1; BChE, 1,095 [834-1,526] U·L-1) compared with healthy volunteers (AChE, 6,693 [5,401-8,020] U·L-1; BChE, 2,645 [2,198-3,478] U·L-1). Patients with viral sepsis with SAE during their ICU stay had lower AChE activity three days after sepsis onset than patients without SAE (4,249 [3,798-5,351] U·L-1 vs 5,544 [4,124-6,461] U·L-1). Butyrylcholinesterase activity seven days after sepsis onset was lower in patients with viral sepsis who died in the ICU than in surviving patients (1,427 [865-2,181] U·L-1 vs 2,122 [1,571-2,787] U·L-1). CONCLUSION: Cholinesterase activities may be relevant prognostic markers for the occurrence of SAE and mortality in the ICU in patients with viral sepsis. STUDY REGISTRATION: This study constitutes an analysis of data from the ongoing studies ICROS (NCT03620409, first submitted 15 May 2018) and ICROVID (DRKS00024162, first submitted 9 February 2021).

RéSUMé: OBJECTIF: Certaines données probantes soutiennent que le déséquilibre cholinergique secondaire à la neuroinflammation joue un rôle dans la physiopathologie de l'encéphalopathie associée au sepsis (EAS). Les activités de l'acétylcholinestérase (AChE) et de la butyrylcholinestérase (BChE) sanguines ont été proposées comme paramètres de substitution de la fonction cholinergique du système nerveux central. Le sepsis viral est associé à une inflammation systémique et il a été rapporté que la BChE possédait une valeur pronostique dans une petite cohorte atteinte de COVID-19. Néanmoins, la valeur pronostique de l'AChE chez les patient·es atteint·es de sepsis viral reste incertaine. MéTHODE: Nous avons étudié le rôle des activités de l'AChE et de la BChE en tant que biomarqueurs pronostiques de l'EAS et de la mortalité chez les patient·es atteint·es de sepsis viral vs non viral recruté·es dans deux études de cohorte prospectives. Nous avons quantifié les activités de l'AChE et de la BChE dans le sang total de patient·es à deux moments de la phase aiguë du sepsis viral (N = 108) et les avons comparées aux activités chez les patient·es atteint·es de sepsis non viral (N = 117) et chez des volontaires sain·es (N = 81). Les patient·es ont été observé·es jusqu'à leur sortie de l'unité de soins intensifs (USI). RéSULTATS: Trois jours après l'apparition du sepsis, les taux médians [écart interquartile] d'AChE et BChE étaient réduits tant chez la patientèle atteinte de sepsis viral (AChE, 5105 [4010­6250] U·L−1; BChE, 1943 [1393­2468] U·L−1) et de sepsis non viral (AChE, 4424 [3630­5055] U·L−1; BChE, 1095 [834­1526] U·L−1) par rapport aux volontaires sain·es (AChE, 6693 [5401­8020] U·L−1; BChE, 2645 [2198­3478] U·L−1). Les patient·es atteint·es de sepsis viral avec EAS pendant leur séjour aux soins intensifs avaient une activité AChE plus faible trois jours après l'apparition du sepsis que les personnes sans EAS (4249 [3798­5351] U·L−1 vs 5544 [4124­6461] U·L−1). L'activité de la butyrylcholinestérase sept jours après l'apparition du sepsis était plus faible chez les patient·es atteint·es de sepsis viral décédé·es à l'USI que chez les personnes ayant survécu (1427 [865­2181] U·L-1 vs 2122 [1571­2787] U·L-1). CONCLUSION: Les activités des cholinestérases pourraient constituer des marqueurs pronostiques pertinents pour la survenue d'EAS et la mortalité en soins intensifs chez la patientèle atteinte de sepsis viral. ENREGISTREMENT DE L'éTUDE: Cette étude constitue une analyse des données des études en cours ICROS (NCT03620409, première soumission le 15 mai 2018) et ICROVID (DRKS00024162, première soumission le 9 février 2021).

Enhancing predictions with a stacking ensemble model for ICU mortality risk in patients with sepsis-associated encephalopathy.

OBJECTIVE: We identified predictive factors and developed a novel machine learning (ML) model for predicting mortality risk in patients with sepsis-associated encephalopathy (SAE). METHODS: In this retrospective cohort study, data from the Medical Information Mart for Intensive Care IV (MIMIC-IV) and eICU Collaborative Research Database were used for model development and external validation. The primary outcome was the in-hospital mortality rate among patients with SAE; the observed in-hospital mortality rate was 14.74% (MIMIC IV: 1112, eICU: 594). Using the least absolute shrinkage and selection operator (LASSO), we built nine ML models and a stacking ensemble model and determined the optimal model based on the area under the receiver operating characteristic curve (AUC). We used the Shapley additive explanations (SHAP) algorithm to determine the optimal model. RESULTS: The study included 9943 patients. LASSO identified 15 variables. The stacking ensemble model achieved the highest AUC on the test set (0.807) and 0.671 on external validation. SHAP analysis highlighted Glasgow Coma Scale (GCS) and age as key variables. The model (https://sic1.shinyapps.io/SSAAEE/) can predict in-hospital mortality risk for patients with SAE. CONCLUSIONS: We developed a stacked ensemble model with enhanced generalization capabilities using novel data to predict mortality risk in patients with SAE.

Targeting blood-brain barrier for sepsis-associated encephalopathy: Regulation of immune cells and ncRNAs.

Sepsis causes significant morbidity and mortality worldwide, most surviving patients show acute or chronic mental disorders, which are known as sepsis-associated encephalopathy (SAE). SAE involves many pathological processes, including the blood-brain barrier (BBB) damage. The BBB is located at the interface between the central nervous system and the surrounding environment, which protects the central nervous system (CNS) from the invasion of exogenous molecules, harmful substances or microorganisms in the blood. Recently, a growing number of studies have indicated that the BBB destruction was involved in SAE and played an important role in SAE-induced brain injury. In the present review, we firstly reveal the pathological processes of SAE such as the neurotransmitter disorders, oxidative stress, immune dysfunction and BBB destruction. Moreover, we introduce the structure of BBB, and describe the immune cells including microglia and astrocytes that participate in the BBB destruction after SAE. Furthermore, in view of the current research on non-coding RNAs (ncRNAs), we explain the regulatory mechanism of ncRNAs including long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) on BBB in the processes of SAE. Finally, we propose some challenges and perspectives of regulating BBB functions in SAE. Hence, on the basis of these effects, both immune cells and ncRNAs may be developed as therapeutic targets to protect BBB for SAE patients.

Serotonergic neurotransmission mediated cognitive dysfunction in two mouse models of sepsis-associated encephalopathy.

BACKGROUND: Patients with sepsis-associated encephalopathy (SAE) often exhibit cognitive impairments. Despite this, the underlying mechanisms of SAE remain largely unexplored. Here, we explored the role of serotonergic neurotransmission in cognitive dysfunction of two mouse models of SAE. METHODS: The mouse models of SAE were established by injection of lipopolysaccharide (LPS, 10 mg/kg, intraperitoneal) and cecal ligation puncture (CLP) respectively. Barnes maze, new object recognition test and open field test were used to evaluate the effects of fluoxetine (selective serotonin reuptake inhibitor) and cyproheptadine (nonselective 5-HT2 receptor antagonist) on cognition and motor activity of mice. Additionally, WAY100635 (5-HT1A receptor antagonist) was co-administered with fluoxetine to explore the mechanism underlying effect of fluoxetine on cognitive impairments of SAE. Enzyme-linked immunosorbent assay (ELISA) was performed to determine 5-HT levels in hippocampus, brainstem and frontal lobe of experimental groups. RESULTS: Both LPS-induced sepsis and CLP induced sepsis resulted in a notable learning deficit. Fluoxetine ameliorated, while cyproheptadine aggravated, cognitive impairment in two classic mouse models of SAE. The cognition-enhancing effect of fluoxetine is reversed by WAY100635. Decreased 5-HT levels in hippocampus, brainstem and frontal lobe were observed in LPS septic model and CLP septic model. Notably, both fluoxetine and cyproheptadine significantly increased 5-HT levels in those brain regions in LPS septic model. Additionally, fluoxetine significantly increased 5-HT levels in frontal lobe of CLP septic model. CONCLUSIONS: Our study demonstrated that serotonergic neurotransmission plays a significant role in mechanisms underlying cognitive impairment in SAE. These findings contribute to identification of novel targets to prevent and arrest cognitive impairment in SAE.

Sepsis-associated encephalopathy: Autophagy and miRNAs regulate microglial activation.

Sepsis-associated encephalopathy (SAE) describes diffuse or multifocal cerebral dysfunction caused by the systemic inflammatory response to sepsis. SAE is a common neurological complication in patients in the middle and late stages of sepsis in the intensive care unit. Microglia, resident macrophages of the central nervous system, phagocytose small numbers of neuronal cells and apoptotic cells, among other cells, to maintain the dynamic balance of the brain's internal environment. The neuroinflammatory response induced by activated microglia plays a central role in the pathogenesis of various central nervous system diseases. In this paper, we systematically describe the functions and phenotypes of microglia, summarize how microglia mediate neuroinflammation and contribute to the occurrence and development of SAE, and discuss recent progress in autophagy- and microRNA-mediated regulation of microglial activation to provide a theoretical basis for the prevention and treatment of SAE and identify related therapeutic targets.

[Construction and analysis of early warning and prediction model for risk factors of sepsis-associated encephalopathy].

OBJECTIVE: To investigate the epidemiological characteristics of sepsis-associated encephalopathy (SAE) in patients with sepsis, analyze its risk factors and build a prediction model, which provides evidence for early clinical identification of SAE patients and improvement of clinical outcomes. METHODS: A retrospective observational study was conducted. Sepsis patients admitted to the critical care medical center of the First Affiliated Hospital of Xinjiang Medical University from February 2022 to February 2023 were enrolled. According to whether SAE occurred, the patients were divided into sepsis group and SAE group. The 24 patients without sepsis in the same period were used as controls (non-sepsis group). Demographic data, relevant scores and laboratory test indicators at admission to intensive care unit (ICU), and prognostic indicators were collected. Univariate and multivariate Logistic regression analysis was used to analyze the risk factors for sepsis and SAE. Receiver operator characteristic curve (ROC curve) was drawn. The predictive value of each risk factor for sepsis and SAE. RESULTS: A total of 130 patients with sepsis were included, of which 52 had SAE, and the incidence of SAE was 40.00%. There were significant differences in the length of ICU stay and total length of stay among all groups, while there were no significant differences in hospitalization cost and mechanical ventilation time. Multivariate Logistic regression analysis showed that pulmonary infection [odds ratio (OR) = 46.817, 95% confidence interval (95%CI) was 5.624-389.757, P = 0.000], acute physiology and chronic health evaluation II (APACHE II: OR = 1.184, 95%CI was 1.032-1.358, P = 0.016), sequential organ failure assessment (SOFA: OR = 9.717, 95%CI was 2.618-36.068, P = 0.001), Charson comorbidity index (CCI: OR = 4.836, 95%CI was 1.860-12.577, P = 0.001), hemoglobin (Hb: OR = 0.893, 95%CI was 0.826-0.966, P = 0.005), glutamyltranspeptidase (OR = 1.026, 95%CI was 1.008-1.045, P = 0.006) were independent risk factors for sepsis in ICU patients. Pulmonary infection (OR = 28.795, 95%CI was 3.296-251.553, P = 0.002), APACHE II score (OR = 1.273, 95%CI was 1.104-1.467, P = 0.001), SOFA score (OR = 8.670, 95%CI was 2.330-32.261, P = 0.001), CCI (OR = 5.141, 95%CI was 1.961-13.475, P = 0.001), Hb (OR = 0.922, 95%CI was 0.857-0.993, P = 0.031), glutamyltranspeptidase (OR = 1.020, 95%CI was 1.002-1.038, P = 0.030) were independent risk factors for SAE in sepsis patients. ROC curve analysis showed that the area under the curve (AUC) of pulmonary infection, APACHE II score, SOFA score, CCI, Hb, and glutamyltranspeptidase for predicting sepsis were 0.792, 0.728, 0.987, 0.933, 0.720, and 0.699, respectively; the AUC of the combined prediction of the above 6 variables for sepsis was 1.000, with a sensitivity of 100% and a specificity of 100%. The AUC predicted by pulmonary infection, APACHE II score, SOFA score, CCI, and Hb for SAE were 0.776, 0.810, 0.907, 0.917, and 0.758, respectively; the AUC of the combined prediction of the above 5 variables for SAE was 0.975, with a sensitivity of 97.3% and a specificity of 93.1%. CONCLUSIONS: Sepsis is more severe when accompanied by encephalopathy. Pulmonary infection, Hb, APACHE II score, SOFA score and CCI were independent risk factors of SAE. The combination of the above five indicators has good predictive value for early screening and prevention of the disease.

Combining biomarkers of BNIP3 L, S100B, NSE, and accessible measures to predict sepsis-associated encephalopathy: a prospective observational study.

BACKGROUND: Accurate identification of delirium in sepsis patients is crucial for guiding clinical diagnosis and treatment. However, there are no accurate biomarkers and indicators at present. We aimed to identify which combinations of cognitive impairment-related biomarkers and other easily accessible assessments best predict delirium in sepsis patients. METHODS: One hundred and one sepsis patients were enrolled in a prospective study cohort. S100B, NSE, and BNIP3 L biomarkers were detected in plasma and cerebrospinal fluid and patients' optic nerve sheath diameter (ONSD). The optimal biomarkers identified by Logistic regression are combined with other factors such as ONSD to filter out the perfect model to predict delirium in sepsis patients through Logistic regression, Naïve Bayes, decision tree, and neural network models. MAIN RESULTS: Among all biomarkers, compared with BNIP3 L (AUC = .706, 95% CI = .597-.815) and NSE (AUC = .711, 95% CI = .609-.813) in cerebrospinal fluid, plasma S100B (AUC = .729, 95% CI = .626-.832) had the best discrimination performance for delirium in sepsis patients. Logistic regression analysis showed that the combination of cerebrospinal fluid BNIP3 L with plasma S100B, ONSD, neutrophils, and age provided the best discrimination to cognitive impairment in sepsis patients (accuracy = .901, specificity = .923, sensitivity = .911), which was better than Naïve Bayes, decision tree, and neural network models. Neutrophils, ONSD, and cerebrospinal fluid BNIP3 L were consistently the major contributors in a few models. CONCLUSIONS: The logistic regression showed that the combination model was strongly correlated with cognitive dysfunction in sepsis patients.

Activating astrocytic α2A adrenoceptors in hippocampus reduces glutamate toxicity to attenuate sepsis-associated encephalopathy in mice.

BACKGROUND: Glutamate metabolism disorder is an important mechanism of sepsis-associated encephalopathy (SAE). Astrocytes regulate glutamate metabolism. In septic mice, α2A adrenoceptor (α2A-AR) activation in the central nervous system provides neuroprotection. α2A-ARs are expressed abundantly in hippocampal astrocytes. This study was performed to determine whether hippocampal astrocytic α2A-AR activation confers neuroprotection against SAE and whether this protective effect is astrocyte specific and achieved by the modulation of glutamate metabolism. METHODS: Male C57BL/6 mice with and without α2A-AR knockdown were subjected to cecal ligation and puncture (CLP). They were treated with intrahippocampal guanfacine (an α2A-AR agonist) or intraperitoneal dexmedetomidine in the presence or absence of dihydrokainic acid [DHK; a glutamate transporter 1 (GLT-1) antagonist] and/or UCPH-101 [a glutamate/aspartate transporter (GLAST) antagonist]. Hippocampal tissue was collected for the measurement of astrocyte reactivity, GLT-1 and GLAST expression, and glutamate receptor subunit 2B (GluN2B) phosphorylation. In vivo real-time extracellular glutamate concentrations in the hippocampus were measured by ultra-performance liquid chromatography tandem mass spectrometry combined with microdialysis, and in vivo real-time hippocampal glutamatergic neuron excitability was assessed by calcium imaging. The mice were subjected to the Barnes maze and fear conditioning tests to assess their learning and memory. Golgi staining was performed to assess changes in the hippocampal synaptic structure. In vitro, primary astrocytes with and without α2A-AR knockdown were stimulated with lipopolysaccharide (LPS) and treated with guanfacine or dexmedetomidine in the presence or absence of 8-bromo- cyclic adenosine monophosphate (8-Br-cAMP, a cAMP analog). LPS-treated primary and BV2 microglia were also treated with guanfacine or dexmedetomidine. Astrocyte reactivity, PKA catalytic subunit, GLT-1 an GLAST expression were determined in primary astrocytes. Interleukin-1ß, interleukin-6 and tumor necrosis factor-alpha in the medium of microglia culture were measured. RESULTS: CLP induced synaptic injury, impaired neurocognitive function, increased astrocyte reactivity and reduced GLT-1 and GLAST expression in the hippocampus of mice. The extracellular glutamate concentration, phosphorylation of GluN2B at Tyr-1472 and glutamatergic neuron excitability in the hippocampus were increased in the hippocampus of septic mice. Intraperitoneal dexmedetomidine or intrahippocampal guanfacine administration attenuated these effects. Hippocampal astrocytes expressed abundant α2A-ARs; expression was also detected in neurons but not microglia. Specific knockdown of α2A-ARs in hippocampal astrocytes and simultaneous intrahippocampal DHK and UCPH-101 administration blocked the neuroprotective effects of dexmedetomidine and guanfacine. Intrahippocampal administration of DHK or UCPH-101 alone had no such effect. In vitro, guanfacine or dexmedetomidine inhibited astrocyte reactivity, reduced PKA catalytic subunit expression, and increased GLT-1 and GLAST expression in primary astrocytes but not in primary astrocytes that received α2A-AR knockdown or were treated with 8-Br-cAMP. Guanfacine or dexmedetomidine inhibited microglial reactivity in BV2 but not primary microglia. CONCLUSIONS: Our results suggest that neurocognitive protection against SAE after hippocampal α2A-AR activation is astrocyte specific. This protection may involve the inhibition of astrocyte reactivity and alleviation of glutamate neurotoxicity, thereby reducing synaptic injury. The cAMP/protein kinase A (PKA) signaling pathway is a potential cellular mechanism by which activating α2A-AR modulates astrocytic function.

4-PBA exerts brain-protective effects against sepsis-associated encephalopathy in a mouse model of sepsis.

BACKGROUND: Neuroinflammation assumes a pivotal role in both the etiological underpinnings and the dynamic progression of sepsis-associated encephalopathy (SAE). The occurrence of cognitive deficits with SAE is associated with neuroinflammation. 4-phenyl butyrate (4-PBA) may control inflammation by inhibiting endoplasmic reticulum stress (ERS). The primary objective of this investigation is to scrutinize the effectiveness of 4-PBA in mitigating neuroinflammation induced by lipopolysaccharides (LPS) and its consequent impact on cognitive function decline. METHODS: LPS-injected mice with SAE and LPS-treated BV2 cell were established to serve as experimental paradigms, both contributing to the investigative framework of the study. Cognitive functions were assessed by behavioral tests. Hippocampal neuronal damage was assessed using Golgi staining and Nissl staining. Quantitative PCR assay and immunofluorescence were used to analyze neuroinflammation. Mitochondrial function was examined using transmission electron microscopy. Protein expression analysis was conducted through the application of western blotting methodology, serving as the investigative approach to elucidate molecular signatures in the experimental framework. Endoplasmic reticulum and mitochondrial calcium flow were detected using flow cytometry. To delve deeper into the mechanistic intricacies, the administration of 4µ8c was employed to selectively impede the IRE1α/Xbp1s pathway, constituting a strategic intervention aimed at elucidating underlying regulatory processes. RESULT: Expression levels of ERS-related proteins exhibited a significant upregulation in hippocampal tissues of LPS-treated mice when compared to wild-type (WT) counterparts. The administration of 4-PBA notably ameliorated memory deficits in LPS-treated mice. Furthermore, 4-PBA treatment was found to alleviate oxidative stress and neuroinflammation. Mechanistically, the IRE1α/Xbp1s-Ca2+ signaling pathway played a crucial role in mediating the beneficial effects of mitigating oxidative stress and maintaining mitochondrial calcium homeostasis, with inhibition of the IRE-related pathway displaying opposing effects. CONCLUSION: Our results suggest that administration of 4-PBA treatment significantly attenuates ERS, alleviates cognitive decline, reduces inflammatory damage, and restores mitochondrial dynamics via the IRE1α/Xbp1s-Ca2+-associated pathway, which provides a new potential therapeutic approach to SAE.

Identifying hub genes of sepsis-associated and hepatic encephalopathies based on bioinformatic analysis-focus on the two common encephalopathies of septic cirrhotic patients in ICU.

BACKGROUND: In the ICU ward, septic cirrhotic patients are susceptible to suffering from sepsis-associated encephalopathy and/or hepatic encephalopathy, which are two common neurological complications in such patients. However, the mutual pathogenesis between sepsis-associated and hepatic encephalopathies remains unclear. We aimed to identify the mutual hub genes, explore effective diagnostic biomarkers and therapeutic targets for the two common encephalopathies and provide novel, promising insights into the clinical management of such septic cirrhotic patients. METHODS: The precious human post-mortem cerebral tissues were deprived of the GSE135838, GSE57193, and GSE41919 datasets, downloaded from the Gene Expression Omnibus database. Furthermore, we identified differentially expressed genes and screened hub genes with weighted gene co-expression network analysis. The hub genes were then subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway functional enrichment analyses, and protein-protein interaction networks were constructed. Receiver operating characteristic curves and correlation analyses were set up for the hub genes. Finally, we explored principal and common signaling pathways by using Gene Set Enrichment Analysis and the association between the hub genes and immune cell subtype distribution by using CIBERSORT algorithm. RESULTS: We identified seven hub genes-GPR4, SOCS3, BAG3, ZFP36, CDKN1A, ADAMTS9, and GADD45B-by using differentially expressed gene analysis and weighted gene co-expression network analysis method. The AUCs of these genes were all greater than 0.7 in the receiver operating characteristic curves analysis. The Gene Set Enrichment Analysis results demonstrated that mutual signaling pathways were mainly enriched in hypoxia and inflammatory response. CIBERSORT indicated that these seven hub genes were closely related to innate and adaptive immune cells. CONCLUSIONS: We identified seven hub genes with promising diagnostic value and therapeutic targets in septic cirrhotic patients with sepsis-associated encephalopathy and/or hepatic encephalopathy. Hypoxia, inflammatory, and immunoreaction responses may share the common downstream pathways of the two common encephalopathies, for which earlier recognition and timely intervention are crucial for management of such septic cirrhotic patients in ICU.

Palmatine ameliorated lipopolysaccharide-induced sepsis-associated encephalopathy mice by regulating the microbiota-gut-brain axis.

BACKGROUND: Sepsis-associated encephalopathy (SAE), a common neurological complication from sepsis, is widespread among patients in intensive care unit and is linked to substantial morbidity and mortality rates, thus posing a substantial menace to human health. Due to the intricate nature of SAE's pathogenesis, there remains a dearth of efficacious therapeutic protocols, encompassing pharmaceutical agents and treatment modalities, up until the present time. Palmatine exhibits distinctive benefits in the regulation of inflammation for the improvement of sepsis. Nevertheless, the precise functions of palmatine in treating SAE and its underlying mechanism have yet to be elucidated. PURPOSE: This study aimed to evaluate efficiency of palmatine in SAE mice and its underlying mechanisms. STUDY DESIGN AND METHODS: Behavioral experiments, percent survival rate analysis, histological analysis, immunofluorescence staining, ELISA analysis, were performed to evaluate the efficiency of palmatine in SAE mice. Quantibody® mouse inflammation array glass chip was performed to observe the effects of palmatine on inflammation storm in SAE mice. Real-time quantitative and western blotting analyzes were employed to examine the expression of relevant targets in the Notch1/nuclear factor-kappa B (NF-κB) pathway. Finally, brain tissues metabolomics-based analyzes were performed to detect the differentially expressed metabolites and metabolic pathways. The fecal samples were subjected to microbial 16S rRNA analysis and untargeted metabolomics analysis in order to identify the specific flora and metabolites associated with SAE, thereby further investigating the mechanism of palmatine in SAE mice. RESULTS: Our results showed that palmatine significantly improved nerve function, reduced cell apoptosis in brain tissue, and decreased inflammatory cytokine levels in SAE induced-LPS mice. Meanwhile, our results demonstrate the potential of palmatine in modulating key components of the Notch1/NF-κB pathway, enhancing the expression of tight junction proteins, improving intestinal permeability, promoting the growth of beneficial bacteria (such as Lachnospiraceae_NK4A136_group), inhibiting the proliferation of harmful bacteria (such as Escherichia-Shigella), and mitigating metabolic disorders. Ultimately, these observed effects contribute to the therapeutic efficacy of palmatine in treating SAE. CONCLUSION: The findings of our study have provided confirmation regarding the efficacy of palmatine in the treatment of SAE, thereby establishing a solid foundation for further exploration into SAE therapy and the advancement and investigation of palmatine.

Cannabidiol effect on long-term brain alterations in septic rats: Involvement of PPARγ activation.

Sepsis is a life-threatening condition induced by a deregulated host response to infection. Post-sepsis injury includes long-term cognitive impairment, whose neurobiological mechanisms and effective treatment remain unknown. The present study was designed to determine the potential effects of cannabidiol (CBD) in a sepsis-associated encephalopathy (SAE) model and explore if peroxisome proliferator activated receptor gamma (PPARγ) is the putative mechanism underpinning the beneficial effects. SAE was induced in Wistar rats by cecal ligation and puncture (CLP) or sham (control). CLP rats received vehicle, CBD (10 mg/kg), PPARγ inhibitor (GW9662 - 1 mg/kg), or GW9662 (1 mg/kg) + CBD (10 mg/kg) intraperitoneally for ten days. During this period, the survival rate was recorded, and at the end of 10 days, a memory test was performed, and the prefrontal cortex and hippocampus were removed to verify brain-derived neurotrophic factor (BDNF), cytokines (IL-1ß, IL-6 and IL-10), myeloperoxidase activity, nitrite nitrate concentration, and lipid and protein carbonylation and catalase activity. Septic rats presented cognitive decline and an increase in mortality following CLP. Only CBD alone improved the cognitive impairment, which was accompanied by restoration of BDNF, reduced neuroinflammation, and oxidative stress, mainly in the hippocampus. This study shows that CLP induces an increase in brain damage and CBD has neuroprotective effects on memory impairment and neurotrophins, as well as against neuroinflammation and oxidative stress, and is mediated by PPARγ activation.

Enhanced meningeal lymphatic drainage ameliorates lipopolysaccharide-induced brain injury in aged mice.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is an acute cerebral dysfunction caused by sepsis. Neuroinflammation induced by sepsis is considered a potential mechanism of SAE; however, very little is known about the role of the meningeal lymphatic system in SAE. METHODS: Sepsis was established in male C57BL/6J mice by intraperitoneal injection of 5 mg/kg lipopolysaccharide, and the function of meningeal lymphatic drainage was assessed. Adeno-associated virus 1-vascular endothelial growth factor C (AAV1-VEGF-C) was injected into the cisterna magna to induce meningeal lymphangiogenesis. Ligation of deep cervical lymph nodes (dCLNs) was performed to induce pre-existing meningeal lymphatic dysfunction. Cognitive function was evaluated by a fear conditioning test, and inflammatory factors were detected by enzyme-linked immunosorbent assay. RESULTS: The aged mice with SAE showed a significant decrease in the drainage of OVA-647 into the dCLNs and the coverage of the Lyve-1 in the meningeal lymphatic, indicating that sepsis impaired meningeal lymphatic drainage and morphology. The meningeal lymphatic function of aged mice was more vulnerable to sepsis in comparison to young mice. Sepsis also decreased the protein levels of caspase-3 and PSD95, which was accompanied by reductions in the activity of hippocampal neurons. Microglia were significantly activated in the hippocampus of SAE mice, which was accompanied by an increase in neuroinflammation, as indicated by increases in interleukin-1 beta, interleukin-6 and Iba1 expression. Cognitive function was impaired in aged mice with SAE. However, the injection of AAV1-VEGF-C significantly increased coverage in the lymphatic system and tracer dye uptake in dCLNs, suggesting that AAV1-VEGF-C promotes meningeal lymphangiogenesis and drainage. Furthermore, AAV1-VEGF-C reduced microglial activation and neuroinflammation and improved cognitive dysfunction. Improvement of meningeal lymphatics also reduced sepsis-induced expression of disease-associated genes in aged mice. Pre-existing lymphatic dysfunction by ligating bilateral dCLNs aggravated sepsis-induced neuroinflammation and cognitive impairment. CONCLUSION: The meningeal lymphatic drainage is damaged in sepsis, and pre-existing defects in this drainage system exacerbate SAE-induced neuroinflammation and cognitive dysfunction. Promoting meningeal lymphatic drainage improves SAE. Manipulation of meningeal lymphangiogenesis could be a new strategy for the treatment of SAE.

Brain-Derived Exosomal miRNA Profiles upon Experimental SAE Rats and Their Comparison with Peripheral Exosomes.

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction secondary to body infection without overt central nervous system infection. Dysregulation of miRNA expression in the transcriptome can spread through RNA transfer in exosomes, providing an early signal of impending neuropathological changes in the brain. Here, we comprehensively analyzed brain-derived exosomal miRNA profiles in SAE rats (n = 3) and controls (n = 3). We further verified the differential expression and correlation of brain tissue, cerebrospinal fluid, and plasma exosomal miRNAs in SAE rats. High-throughput sequencing of brain-derived exosomal miRNAs identified 101 differentially expressed miRNAs, of which 16 were downregulated and 85 were upregulated. Four exosomal miRNAs (miR-127-3p, miR-423-3p, mR-378b, and miR-106-3p) were differentially expressed and correlated in the brain tissue, cerebrospinal fluid, and plasma, revealing the potential use of miRNAs as SAE liquid brain biopsies. Understanding exosomal miRNA profiles in SAE brain tissue and exploring the correlation with peripheral exosomal miRNA can contribute to a comprehensive understanding of miRNA changes in the SAE pathological process and provide the possibility of establishing early diagnostic assays.

Effect of esketamine pretreatment on acute sepsis-associated encephalopathy.

PURPOSE: Esketamine, the S(+) enantiomer of ketamine, exhibits good anesthetic efficacy and controllability; however, its potential clinical applications, particularly in sepsis-associated encephalopathy (SAE), remain underexplored. SAE involves the development of diffuse brain dysfunction after sepsis, leading to markedly increased sepsis-related disability and mortality. In this study, we investigated the effects of esketamine pretreatment on acute SAE. METHODS: Mice were randomly divided into four groups: control (C, n = 22), acute SAE (L, n = 22), esketamine pretreatment + acute SAE (EL, n = 22), and nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor (ML385) + esketamine pretreatment + acute SAE (N + EL, n = 22). Acute SAE was established using intraperitoneal (i.p.) injection of lipopolysaccharide (LPS; 10 mg/kg), while controls received equal amounts of saline. The EL group received daily i.p. injections of esketamine (10 mg/kg) for 5 consecutive days, followed by LPS on day 6. The N + EL group received i.p. injections of ML385 (30 mg/kg) 1 h before esketamine pretreatment. The remainder of treatment followed the same protocol as the EL group. Behavioral tests were performed 24 h post-LPS injection, and whole blood and brain tissues were collected for further analysis. RESULTS: Esketamine improved sepsis symptoms, 7-day survival, and spatial cognitive impairment, without altering locomotor activity. Moreover, esketamine reversed the LPS-induced increase in serum S100 calcium-binding protein ß and neuron-specific enolase levels and reduced hippocampal neuroinflammation, oxidative stress, and neuronal apoptosis in the EL group. However, these neuroprotective effects of esketamine were reversed by ML385. CONCLUSION: The results of our study suggest that esketamine pretreatment mitigates acute SAE, highlighting the involvement of the Nrf2/heme oxygenase-1 pathway in mediating its neuroprotective effects.

Potential of piperine for neuroprotection in sepsis-associated encephalopathy.

AIMS: Sepsis-associated encephalopathy (SAE) is a common complication that increases mortality and leads to long-term cognitive impairment in sepsis survivors. However, no specific or effective therapy has been identified for this complication. Piperine is an alkaloid known for its anti-inflammatory, antioxidant, and neuroprotective properties, which are important characteristics for treatment of SAE. The objective of this study was to evaluate the neuroprotective effect of piperine on SAE in C57BL/6 mice that underwent cecum ligation and perforation surgery (CLP). MAIN METHODS: C57BL/6 male mice were randomly assigned to groups that underwent SHAM surgery or CLP. Mice in the CLP group were treated with piperine at doses of 20 or 40 mg/kg for short- (5 days) or long-term (10 days) periods after CLP. KEY FINDINGS: Our results revealed that untreated septic animals exhibited increased concentrations of IL-6, TNF, VEGF, MMP-9, TBARS, and NLRP3, and decreased levels of BDNF, sulfhydryl groups, and catalase in the short term. Additionally, the levels of carbonylated proteins and degenerated neuronal cells were increased at both time points. Furthermore, short-term and visuospatial memories were impaired. Piperine treatment reduced MMP-9 activity in the short term and decreased the levels of carbonylated proteins and degenerated neuronal cells in the long term. It also lowered IL-6 and TBARS levels at both time points evaluated. Moreover, piperine increased short-term catalase and long-term BDNF factor levels and improved memory at both time points. SIGNIFICANCE: In conclusion, our data demonstrate that piperine exerts a neuroprotective effect on SAE in animals that have undergone CLP.