Cecal Ligation And Puncture Model Research Articles

Berberine alleviates neuroinflammation by downregulating NFκB/LCN2 pathway in sepsis-associated encephalopathy: network pharmacology, bioinformatics, and experimental validation

BackgroundSepsis refers to a systemic inflammatory response caused by infection, involving multiple organs. Sepsis-associated encephalopathy (SAE), as one of the most common complications in patients with severe sepsis, refers to the diffuse brain dysfunction caused by sepsis without central nervous system infection. However, there is no clear diagnostic criteria and lack of specific diagnostic markers. MethodsThe main active ingredients of coptidis rhizoma(CR) were identified from TCMSP and SwissADME databases. SwissTargetPrediction and PharmMapper databases were used to obtain targets of CR. OMIM, DisGeNET and Genecards databases were used to explore targets of SAE. Limma differential analysis was used to identify the differential expressed genes(DEGs) in GSE167610 and GSE198861 datasets. WGCNA was used to identify feature module. GO and KEGG enrichment analysis were performed using Metascape, DAVID and STRING databases. The PPI network was constructed by STRING database and analyzed by Cytoscape software. AutoDock and PyMOL software were used for molecular docking and visualization. Cecal ligation and puncture(CLP) was used to construct a mouse model of SAE, and the core targets were verified in vivo experiments. Results277 common targets were identified by taking the intersection of 4730 targets related to SAE and 509 targets of 9 main active ingredients of CR. 52 common DEGs were mined from GSE167610 and GSE198861 datasets. Among the 25,864 DEGs in GSE198861, LCN2 showed the most significant difference (logFC = 6.9). GO and KEGG enrichment analysis showed that these 52 DEGs were closely related to “inflammatory response” and “innate immunity”. A network containing 38 genes was obtained by PPI analysis, among which LCN2 ranked the first in Degree value. Molecular docking results showed that berberine had a well binding affinity with LCN2. Animal experiments results showed that berberine could inhibit the high expression of LCN2,S100A9 and TGM2 induced by CLP in the hippocampus of mice, as well as the high expression of inflammatory factors (TNFα, IL-6 and IL-1β). In addition, berberine might reduce inflammation and neuronal cell death by partially inhibiting NFκB/LCN2 pathway in the hippocampus of CLP models, thereby alleviating SAE. ConclusionOverall, Berberine may exert anti-inflammatory effects through multi-ingredients, multi-targets and multi-pathways to partially rescue neuronal death and alleviate SAE.

The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis across Murine Models.

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).

Mesenchymal stem cells alleviate sepsis-induced acute lung injury by blocking neutrophil extracellular traps formation and inhibiting ferroptosis in rats.

Acute lung injury (ALI) is one of the most serious complications of sepsis, characterized by high morbidity and mortality rates. Ferroptosis has recently been reported to play an essential role in sepsis-induced ALI. Excessive neutrophil extracellular traps (NETs) formation induces exacerbated inflammation and is crucial to the development of ALI. In this study, we explored the effects of ferroptosis and NETs and observed the therapeutic function of mesenchymal stem cells (MSCs) on sepsis-induced ALI. First, we produced a cecal ligation and puncture (CLP) model of sepsis in rats. Ferrostain-1 and DNase-1 were used to inhibit ferroptosis and NETs formation separately, to confirm their effects on sepsis-induced ALI. Next, U0126 was applied to suppress the MEK/ERK signaling pathway, which is considered to be vital to NETs formation. Finally, the therapeutic effect of MSCs was observed on CLP models. The results demonstrated that both ferrostain-1 and DNase-1 application could improve sepsis-induced ALI. DNase-1 inhibited ferroptosis significantly in lung tissues, showing that ferroptosis could be regulated by NETs formation. With the inhibition of the MEK/ERK signaling pathway by U0126, NETs formation and ferroptosis in lung tissues were both reduced, and sepsis-induced ALI was improved. MSCs also had a similar protective effect against sepsis-induced ALI, not only inhibiting MEK/ERK signaling pathway-mediated NETs formation, but also alleviating ferroptosis in lung tissues. We concluded that MSCs could protect against sepsis-induced ALI by suppressing NETs formation and ferroptosis in lung tissues. In this study, we found that NETs formation and ferroptosis were both potential therapeutic targets for the treatment of sepsis-induced ALI, and provided new evidence supporting the clinical application of MSCs in sepsis-induced ALI treatment.

Rapamycin mitigates organ damage by autophagy-mediated NLRP3 inflammasome inactivation in sepsis.

Autophagy activation can alleviate sepsis-induced organ injuries. Rapamycin (Rap) has emerged as an autophagy regulator in multiple forms of organ injuries. This study aimed to assess whether Rap protects rats from cecal ligation and puncture (CLP)-induced sepsis through autophagy-mediated inactivation of the NLRP3 inflammasome. Rats were allocated to the sham, CLP, Rap (10 mg/kg), or 3-Methyladenine (3-MA) (15 mg/kg) groups. A rat CLP model was established. The survival of rats and lung wet-to-dry weight ratio in each group was assessed. Blood biochemical indexes and oxidative stress-related factors were analyzed with an automatic biochemical analyzer. The bacterial counts of blood and organs were monitored. The degrees of myeloperoxidase of the ileum, inflammation-related indexes, and pathological changes in the tissues were detected by ELISA and hematoxylin-eosin staining. The levels of NLRP3 inflammasome and autophagy-related factors were analyzed by Western blot. Rap increased the survival and SOD activity, and repressed ALT, AST, BUN, SCr, MDA, and inflammation-related marker levels in CLP rats, it also restrained the bacterial counts of blood, lung, liver, and kidney in CLP rats; the effects of 3-MA on CLP rats on the above-mentioned indicators were opposite to those of Rap. Additionally, Rap alleviated the pathological injury of the lung, liver, and kidney, which was the opposite to the effect of 3-MA on CLP rats. Furthermore, Rap mitigated the ASC, Pro-caspase 1, and NLRP3 levels and increased the Beclin-1 levels and the LC3II/LC3I ratio in the organ tissues. Collectively, autophagy activation can mitigate organ damage by suppressing the NLRP3 inflammasome in sepsis rats.

A Novel Scoring System for Humane Endpoints in Mice with Cecal Ligation and Puncture-Induced Sepsis.

Animal-based research is essential to the study of sepsis pathophysiology, diagnostics, and therapeutics. However, animal models of sepsis are often associated with high mortality because of the difficulty in predicting imminent death based on premortem assessment of the animals. The use of validated visual scoring would allow researchers to systematically identify humane endpoints but visual approaches require high interobserver agreement for accurate results. The objective of this study was to establish a scoring system for mice undergoing cecal ligation and puncture (CLP)-induced sepsis based on 3 visual parameters: respiratory status, activity and response to stimulus (ASR), and eye appearance, with scores ranging from 0 to 3. In the first study, we evaluated interobserver agreement. Veterinary and investigative staff assessed 283 mice with CLP and had substantial to near-perfect agreement for all 3 parameters as evaluated using weighted Cohen κ statistic. The second study assessed the ability of the scoring system and temperature to predict death. The scoring system and subcutaneous transpond- ers were used to monitor C57BL/6J mice (n = 80, male and female) until death or for 7 days after CLP. Results showed that the scoring system discriminates between surviving (n = 26) and nonsurviving (n = 54) septic mice. The scoring system was accurate in predicting death, with an AUC of 0.8997. The sensitivity and specificity of the ASR parameter were 96% and 92%, respectively, and for the eye parameter were 94% and 73%. A sum of the ASR and eye scores that was 5 or more was also predictive of death. Temperature was a quantitative predictor, with sensitivity and specificity of 93% and 92%, respectively. This scoring system refines the CLP model by allowing identification of humane endpoints and avoidance of spontaneous death.

ADENOSINE INFLUENCES FOXP3 EXPRESSION OF T REGS VIA THE A2AR/CREB PATHWAY IN A MOUSE MODEL OF SEPSIS.

The adenosine concentration and forkhead box protein (Foxp3) expression in T regulatory cells (T regs ) are increased during sepsis. However, the mechanism by which adenosine induces Foxp3 expression is incompletely understood. A cecal ligation and puncture (CLP) model was constructed using C57BL/J mice. The plasma adenosine concentration and Foxp3 expression in splenic T regs were increased consistently for 15 days after sepsis onset. Analysis of the mean fluorescence intensity of Foxp3 and adenosine concentration in the same mice revealed a linear correlation. In the CLP model, adenosine 2a receptor (A2aR) blockade inhibited Foxp3 expression in T regs . In vitro activation of A2aR promoted Foxp3 expression in T regs and facilitated secretion of extracellular vesicles. Transcriptome sequencing revealed that A2aR blockade led to changes in cyclic adenosine monophosphate response element-binding protein (CREB) transcription in T regs in our sepsis model. Use of adenosine or A2aR agonists promoted CREB expression, CREB phosphorylation at S133, T reg expression of Foxp3, and enhanced inhibition of proliferation of cluster of differentiation (CD)4+ lymphocytes. A2aR blockade or inhibition of CREB expression inhibited Foxp3 expression in T regs . In the CLP model, use of CREB inhibitors could inhibit Foxp3 expression and reduce the bacterial load. In summary, adenosine in sepsis promotes CREB phosphorylation via A2aR which, in turn, upregulates Foxp3 expression in T regs .

Common Variables That Influence Sepsis Mortality in Mice.

Sepsis is characterized by a dysregulated host immune response to infection, leading to organ dysfunction and a high risk of death. The cecal ligation and puncture (CLP) mouse model is commonly used to study sepsis, but animal mortality rates vary between different studies. Technical factors and animal characteristics may affect this model in unanticipated ways, and if unaccounted for, may lead to serious biases in study findings. We sought to evaluate whether mouse sex, age, weight, surgeon, season of experiments, and timing of antibiotic administration influenced mortality in the CLP model. We created a comprehensive dataset of C57BL/6J mice that had undergone CLP surgery within our lab during years 2015-2020 from published and unpublished studies. The primary outcome was defined as the time from sepsis induction to death or termination of study (14 days). The Log rank test and Cox regression models were used to analyze the dataset. The study included 119 mice, of which 43% were female, with an average age of 12.6 weeks, an average weight of 25.3 g. 38 (32%) of the animals died. In the unadjusted analyses, experiments performed in the summer and higher weight predicted a higher risk of mortality. In the stratified Cox model by sex, summer season (adjusted hazard ratio [aHR]=5.61, p=0.004) and delayed antibiotic administration (aHR=1.46, p=0.029) were associated with mortality in males, whereas higher weight (aHR=1.52, p=0.005) significantly affected mortality in females. In addition, delayed antibiotic administration (HR=1.42, p=0.025) was associated with mortality in the non-summer seasons, but not in the summer season. In conclusion, some factors specific to sex and season have a significant influence on sepsis mortality in the CLP model. Consideration of these factors along with appropriate group matching or adjusted analysis is critical to minimize variability beyond the experimental conditions within a study.

Abcc8 (sulfonylurea receptor-1) knockout mice exhibit reduced axonal injury, cytotoxic edema and cognitive dysfunction vs. wild-type in a cecal ligation and puncture model of sepsis

Sepsis-associated brain injury (SABI) is characterized by an acute deterioration of mental status resulting in cognitive impairment and acquisition of new and persistent functional limitations in sepsis survivors. Previously, we reported that septic mice had evidence of axonal injury, robust microglial activation, and cytotoxic edema in the cerebral cortex, thalamus, and hippocampus in the absence of blood–brain barrier disruption. A key conceptual advance in the field was identification of sulfonylurea receptor 1 (SUR1), a member of the adenosine triphosphate (ATP)-binding cassette protein superfamily, that associates with the transient receptor potential melastatin 4 (TRPM4) cation channel to play a crucial role in cerebral edema development. Therefore, we hypothesized that knockout (KO) of Abcc8 (Sur1 gene) is associated with a decrease in microglial activation, cerebral edema, and improved neurobehavioral outcomes in a murine cecal ligation and puncture (CLP) model of sepsis. Sepsis was induced in 4–6-week-old Abcc8 KO and wild-type (WT) littermate control male mice by CLP. We used immunohistochemistry to define neuropathology and microglial activation along with parallel studies using magnetic resonance imaging, focusing on cerebral edema on days 1 and 4 after CLP. Abcc8 KO mice exhibited a decrease in axonal injury and cytotoxic edema vs. WT on day 1. Abcc8 KO mice also had decreased microglial activation in the cerebral cortex vs. WT. These findings were associated with improved spatial memory on days 7–8 after CLP. Our study challenges a key concept in sepsis and suggests that brain injury may not occur merely as an extension of systemic inflammation. We advance the field further and demonstrate that deletion of the SUR1 gene ameliorates CNS pathobiology in sepsis including edema, axonal injury, neuroinflammation, and behavioral deficits. Benefits conferred by Abcc8 KO in the murine CLP model warrant studies of pharmacological Abcc8 inhibition as a new potential therapeutic strategy for SABI.

Parthenolide Attenuates Sepsis-Induced Acute Kidney Injury in Rats by Reducing Inflammation.

Sepsis is a common complication of severe trauma, burns, infection, or major surgery. This disease-related end-organ dysfunction results from systemic inflammatory response syndrome (SIRS). Acute kidney damage (AKI), also known as acute renal failure, is one of the most frequent and serious sequelae of sepsis. Nuclear transcription factor-κB (NF-κB) regulates the transcription of inflammation-related genes and operates as a mediator in the immune system. While parthenolide (PTL) has been reported to prevent harmful inflammatory reactions, its effects on sepsis-associated AKI are unknown. The current study investigates the effects of PTL in sepsis-associated AKI using cell and cecal ligation and puncture (CLP) models. Lipopolysaccharide (LPS)-stimulated rat glomerular mesangial cells were treated with 10 μM PTL. Inflammatory mediators, including TNF-α, IL-6, and IL-1β, in the culture supernatants were measured by ELISA, and NF-κB levels were assessed by qPCR. After the generation of the septic CLP model, rats were intraperitoneally injected with 500 g/kg PTL and were euthanized after 72 h. Serum and kidney samples were analyzed. TNF-α, IL-1β, and IL-6 levels were elevated after LPS treatment of rat glomerular mesangial cells (p=0.004, p=0.002, and p=0.004, respectively) but were significantly reduced in the PTL treatment group (p ≤ 0.001, p=0.01, and p ≤ 0.001). NF-κB p65 levels were also increased after LPS treatment in this group and were reduced in the PTL treatment group. PTL treatment also reduced kidney damage after CLP induction, as shown by histological analysis and reductions in the levels of BUN, Cre, KIM-1, and NAGL. CLP-induced kidney inflammation together with increased levels of proinflammatory cytokines and inflammatory-related proteins. The elevated levels of renal TNF-α, IL-6, and IL-1β were downregulated after PTL treatment. The PTL treatment also reduced the CLP-induced activation of NF-κB p65 in the damaged kidneys. PTL reduced inflammation induced by CLP-induced AKI in rat models and LPS-induced damage to glomerular mesangial cells by suppressing NF-κB signaling.

490. Brain-derived neurotrophic factor (BDNF) is a prognostic biomarker and therapeutic target through AMP-activated protein kinase (AMPK) signal pathway for sepsis

Abstract Background Brain-derived neurotrophic factor (BDNF) is a pleiotropic cytokine that not only acts on the central nervous system, but is also implicated in metabolic regulation, development of lung diseases, and support of hematopoiesis. Recently, a study report that the plasma levels of BDNF were independently associated with mortality in critically ill patients. We aimed to evaluate the role of BDNF in sepsis patients and animal sepsis model. Methods Prospectively enrolled adult sepsis patients (n=168) who visited the emergency room (ER) of Korea University Ansan Hospital from January 2017 to December 2018., and healthy volunteers (n=48) were included in the present study. Serum BDNF level was determined and analyzed in septic patients and healthy volunteers. The effects of BDNF on pro-inflammatory cytokines, AMP-activated protein kinase (AMPK) and NF-κB signaling in septic condition of RAW264.7 cells and mouse peritoneal macrophage, and mouse sepsis model by cecal ligation and puncture (CLP). Results Serum BDNF levels (mean) were significantly decreased in sepsis patients compared to healthy controls (11.78ng/ml vs 18.91ng/ml, p< 0.001) and significantly decreased in non-survivors compared to survivors at discharge (3.16ng/ml vs 13.86ng/ml, p< 0.05). BDNF treatment suppressed LPS-induced pro-inflammatory cytokine (TNF-α, iL-6, and iL-1β) in RAW264.7 cells. Although LPS stimulation diminished AMPK phosphorylation, BDNF was able to decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on down stream signaling events, particularly LPS-induced IκBα degradation in RAW cells and peritoneal macrophage. In CLP model, intra-muscular injection of BDNF after CLP significantly decreased serum levels of TNF-α which was increased compared to sham, and significantly improved survival of mouse (p< 0.01). Conclusion Sepsis results in a decreased production of BDNF, which might be a potential prognostic biomarker and therapeutic target through AMPK signal pathway for sepsis. Disclosures All Authors: No reported disclosures.

1235: ABBCC8 (SULFONYLUREA RECEPTOR 1) KO CONFERS NEUROPROTECTION IN SEPSIS-ASSOCIATED BRAIN INJURY

Introduction: Sepsis-Associated Brain Injury (SABI) Is Characterized by an Acute Deterioration of Mental Status Resulting in Cognitive Impairment and Acquisition of New and Persistent Functional Limitations in Sepsis Survivors. Previously, We Reported That Septic Mice Had Evidence of Axonal Injury, Robust Microglial Activation, and Cytotoxic Edema in the Cerebral Cortex, Thalamus, and Hippocampus in the Absence of Blood-Brain Barrier Disruption. In Central Nervous System Injury, Sulfonylurea Receptor 1 (SUR1), a Member of the Adenosine Triphosphate (ATP)-Binding Cassette Protein Superfamily, Associates With the Transient Receptor Potential Melastatin 4 (TRPM4) Cation Channel to Play a Crucial Role in Cerebral Edema Development. Therefore, We Hypothesized That Knockout (KO) of Abcc8 (Sur1 Gene) Is Associated With a Decrease in Microglial Activation, Cerebral Edema, and Improved Neurobehavioral Outcomes in a Murine Cecal Ligation and Puncture (CLP) Model of Sepsis. Methods: Sepsis Was Induced in 4-6-Week-Old Abcc8 KO and Wild-Type (WT) Littermate Control Male Mice by CLP. We Used Immunohistochemistry to Define Microglial Activation Along With Parallel Studies Using Magnetic Resonance Imaging, Focusing on Cerebral Edema on Days 1 and 4 Days After CLP. Results: Abcc8 KO Mice Exhibited a Decrease in Axonal Injury and Cytotoxic Edema vs. WT on Day 1. Abcc8 KO Mice Also Had Decreased Microglial Activation in the Cerebral Cortex vs WT. These Findings Were Associated With Cognitive Improvement on Days 7–8 After CLP. Conclusions: Our Study Challenges a Key Concept in Sepsis and Suggests That Brain Injury Does Not Merely Happen as an Extension of Systemic Inflammation, but Upregulation of the SUR1-TRPM4 Channel in the Brain Directly Contributes to the CNS Pathobiology Previously Demonstrated. Benefits Conferred by Abcc8 KO in the Murine CLP Model Warrant Studies of Pharmacological Abcc8 Inhibition as a New Potential Therapeutic Strategy for SABI.

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

Specificity of Presepsin as a Biomarker of Bacterial Infection in Mouse Sepsis Models

IntroductionSince its discovery in 2002, presepsin (P-SEP) has been reported to be useful in the early diagnosis of sepsis and has been evaluated in many clinical studies. However, as antibodies that bind to mouse P-SEP were previously unavailable, serum P-SEP levels in mice are limited. This study used a P-SEP enzyme-linked immunosorbent assay kit to evaluate the changes in serum P-SEP levels in mouse sepsis models compared with changes in other inflammatory markers and determine whether P-SEP can function as a biomarker specific to bacterial infections. MethodsSepsis was induced in mice via cecal ligation and puncture (CLP), induction with lipopolysaccharide (LPS), and cecal ligation (CL) model was created as a control for the CLP model, following which clinical biomarkers (P-SEP, C-reactive protein, and procalcitonin) were evaluated. ResultsThe 48-h survival rates in the CLP, CL, and LPS-induced sepsis models were 67%, 89%, and 57%, respectively. Serum C-reactive protein levels did not increase in the CLP and CL models within 24 h but significantly increased in the LPS-induced sepsis model. Serum procalcitonin levels increased in the CLP and CL models and especially increased in the LPS-induced sepsis model. In contrast, an increase in serum P-SEP level was found in the CLP model at 6 h compared with those at baseline, the CL, and LPS-induced sepsis models. ConclusionsMouse P-SEP is elevated early in infection and more specific to bacterial infection compared with other biomarkers.

Reactive Microgliosis in Sepsis-Associated and Acute Hepatic Encephalopathies: An Ultrastructural Study.

Sepsis and acute liver failure are associated with severe endogenous intoxication. Microglia, which are the resident immune brain cells, play diverse roles in central nervous system development, surveillance, and defense, as well as contributing to neuroinflammatory reactions. In particular, microglia are fundamental to the pathophysiology of reactive toxic encephalopathies. We analyzed microglial ultrastructure, morphotypes, and phagocytosis in the sensorimotor cortex of cecal ligation and puncture (CLP) and acetaminophen-induced liver failure (AILF) Wistar rats. A CLP model induced a gradual shift of ~50% of surveillant microglia to amoeboid hypertrophic-like and gitter cell-like reactive phenotypes with active phagocytosis and frequent contacts with damaged neurons. In contrast, AILF microglia exhibited amoeboid, rod-like, and hypertrophic-like reactive morphotypes with minimal indications for efficient phagocytosis, and were mostly in contact with edematous astrocytes. Close interactions of reactive microglia with neurons, astrocytes, and blood-brain barrier components reflect an active contribution of these cells to the tissue adaptation and cellular remodeling to toxic brain damage. Partial disability of reactive microglia may affect the integrity and metabolism in all tissue compartments, leading to failure of the compensatory mechanisms in acute endogenous toxic encephalopathies.

Brain aquaporin-4 expression in the rat septic model (immunohistochemical study)

The study aimed to determine aquaporin-4 expression in different brain regions was performed in Wistar rats subjected to cecal ligation and puncture (CLP) septic model. The immunohistochemical study of aquaporin-4 was carried out in the sensorimotor cortex, white matter, hippocampus, thalamus and caudate nucleus/putamen regions between 20 and 38 h after CLP. From the 12th h after CLP all animals showed the progressive impairment of sepsis signs and therefore, 9 rats were euthanized between 20-38 h (“CLP-B”, non-survived); 11 animals survived up to 48 h (con­stituted “CLP-A”, survived). After operation, CLP-B group displayed regionally-specific dynamic increase in aquaporin-4 level in the brain mostly associated with astroglial capillary endfeet: by 23rd h in the cortex – 234.15%, by 24th h in the thalamus –129.47% and hippocampus – 101.36%, by 30th h in the white matter – 135.31% and by 38 h in the caudate/pu­tamen – 92.85%; with the highest increase in cortex: by 3.34 times. Heterogeneous and heterochronous aquaporin-4 elevation among brain regions indicates territories more and less susceptible for systemic toxic exposure in sepsis as well as points to diverse reactive responsiveness of local astroglial populations during specific time-period after CLP. The higher rates of aquaporin-4 in the cortex of non-survived animals in CLP model reflects the importance of aquaporin-4 increase in the mechanisms of sepsis decompensation.

Nicotinamide Mononucleotide Administration Triggers Macrophages Reprogramming and Alleviates Inflammation During Sepsis Induced by Experimental Peritonitis.

Peritonitis and subsequent sepsis lead to high morbidity and mortality in response to uncontrolled systemic inflammation primarily mediated by macrophages. Nicotinamide adenine dinucleotide (NAD+) is an important regulator of oxidative stress and immunoinflammatory responses. However, the effects of NAD+ replenishment during inflammatory activation are still poorly defined. Hence, we investigated whether the administration of β-nicotinamide mononucleotide (β-NMN), a natural biosynthetic precursor of NAD+, could modulate the macrophage phenotype and thereby ameliorate the dysregulated inflammatory response during sepsis. For this purpose, C57BL6 mice were subjected to the cecal ligation and puncture (CLP) model to provoke sepsis or were injected with thioglycolate to induce sterile peritonitis with recruitment and differentiation of macrophages into the inflamed peritoneal cavity. β-NMN was administered for 4 days after CLP and for 3 days post thioglycolate treatment where peritoneal macrophages were subsequently analyzed. In the CLP model, administration of β-NMN decreased bacterial load in blood and reduced clinical signs of distress and mortality during sepsis. These results were supported by transcriptomic analysis of hearts and lungs 24 h post CLP-induction, which revealed that β-NMN downregulated genes controlling the immuno-inflammatory response and upregulated genes involved in bioenergetic metabolism, mitochondria, and autophagy. In the thioglycolate model, a significant increase in the proportion of CD206 macrophages, marker of anti-inflammatory M2 phenotype, was detected on peritoneal exudate macrophages from β-NMN-administered mice. Transcriptomic signature of these macrophages after bacterial stimulation confirmed that β-NMN administration limited the pro-inflammatory M1 phenotype and induced the expression of specific markers of M2 type macrophages. Furthermore, our data show that β-NMN treatment significantly impacts NAD + metabolism. This shift in the macrophage phenotype and metabolism was accompanied by a reduction in phagolysosome acidification and secretion of inflammatory mediators in macrophages from β-NMN-treated mice suggesting a reduced pro-inflammatory activation. In conclusion, administration of β-NMN prevented clinical deterioration and improved survival during sepsis. These effects relied on shifts in the metabolism of organs that face up an increased energy requirement caused by bacterial infection and in innate immunity response, including reprogramming of macrophages from a highly inflammatory phenotype to an anti-inflammatory/pro-resolving profile.

Dihydroartemisinin protects blood-brain barrier permeability during sepsis by inhibiting the transcription factor SNAI1.

Blood–brain barrier (BBB) injury is involved in the pathogenesis of sepsis‐associated encephalopathy. In this study, we used dihydroartemisinin (DHA), a derivative of artemisinin, to treat a cecal ligation and puncture (CLP)‐induced mouse sepsis model and a tumour necrosis factor α (TNF‐α)‐stimulated human cerebral microvessel endothelial cells (hCMEC)/D3 cell line. We found that DHA decreased BBB permeability and increased the expression of the tight junction protein occludin (OCLN) in the CLP model. In hCMEC/D3 cells, DHA decreased TNF‐α‐induced hyperpermeability and increased the expression of OCLN. DHA also repressed SNAI1 expression in the CLP mouse model and in TNF‐α‐stimulated hCMEC/D3 cells. These data suggest that DHA protects BBB permeability during sepsis by stimulating the expression of OCLN, by downregulating the expression of the SNAI1 transcription factor.

Protocol for a Sepsis Model Utilizing Fecal Suspension in Mice: Fecal Suspension Intraperitoneal Injection Model.

BackgroundVarious animal models of sepsis have been developed to optimize sepsis treatment. However, therapeutic agents that were successful in animal models were rarely effective in human clinical trials. The cecal ligation and puncture (CLP) model is currently the gold standard for sepsis studies. However, its limitations include the high variability among researchers and the difficulty in comparing animals with different cecum shapes and sizes. In this study, we established a protocol for the creation of a simple and accessible sepsis rodent model using fecal suspensions that minimized differences in technical effects among researchers and individual differences in animals.MethodsA mouse model of sepsis using fecal suspension intraperitoneal injection (FSI) was created using fresh stool excreted within 24 h. The collected fresh stool was dissolved in saline solution and filtered. The obtained fecal suspension was injected intraperitoneally into the mice. Moreover, fecal suspensions with different concentrations were prepared, and the survival rates were compared among the fecal suspensions for each concentration. To assess the validity of the FSI as a sepsis model, CLP and FSI with similar mortality rates were compared pathologically, physiologically, immunologically, and bacteriologically. Histopathological comparison was evaluated by hematoxylin-eosin and Gram staining of the parenchymal organs. Physiological evaluation was performed by comparing the respiratory rate, body temperature, and blood gas analysis results. Immunological assessment was performed using multiplex analysis. Bacteriological comparisons were performed by culturing ascites fluid.ResultsThe FSI model increased mortality in proportion to the fecal suspension concentration. The mortality rate was reduced with antibiotic administration. In various comparative experiments conducted using the FSI and CLP models, both models showed findings consistent with sepsis. Furthermore, the FSI model showed less variability among the individuals in each test.ConclusionThis is the first detailed and accurate report of a protocol for creating a sepsis model using fecal suspension. The FSI model is a minimally invasive and accessible sepsis rodent model. Its clinical validity as a sepsis model was proven via histological, physiological, microbiological, and immunological evaluation methods. The FSI model minimizes individual differences between mice and helps to conduct accurate studies after the onset of sepsis.

Electrical Stimulation of the Dorsal Motor Nucleus of the Vagus Attenuates Serum Pro‐Inflammatory Cytokine Levels in Murine Models of Endotoxemia and Sepsis

The inflammatory reflex is a vagus nerve‐mediated and brainstem‐integrated physiological mechanism that regulates immunity and inflammation (Annu Rev Immunol, 2018, 36:783). The efferent vagus nerve originates in brainstem nuclei, including the dorsal motor nucleus of the vagus (DMN). Recently, it was shown that optogenetic stimulation of cholinergic DMN neurons inhibits serum TNF levels during endotoxemia (Proc Natl Acad Sci USA, 2020, 117(47):29803). However, the effects of electrical DMN stimulation on cytokine levels in inflammatory conditions have not been previously evaluated. To provide insight, we examined the effect of electrical DMN stimulation in mice with endotoxemia and cecal ligation and puncture (CLP)‐induced polymicrobial sepsis. In the endotoxemia model, 10–12‐week‐old male C57BL/6 mice (n=14‐20 per group) were anesthetized using isoflurane and the head was positioned on a stereotaxic frame. A surgical intervention and a stereotaxic‐guided approach were used to insert a concentric bipolar electrode into the left DMN. Electrical stimulation (50 μA, 30 Hz, 260 μs) or sham stimulation was performed for five mins, followed by lipopolysaccharide (LPS, 0.5 mg/kg) i.p. administration. The mice were euthanized 90 mins after LPS and blood (via cardiac puncture) was collected and processed for cytokine analysis. In the CLP model, DMN stimulation or sham stimulation was performed as described above immediately after subjecting mice (n=14‐20 per group) to CLP, i.e., following a midline laparotomy in anesthetised mice, the cecum was isolated and the distal end was ligated and punctured. 24h later, the mice were euthanised and blood was collected and processed for analysis. In mice with endotoxemia, electrical DMN stimulation significantly decreased serum TNF (p = 0.0069) and IL‐6 (p = 0.0427) levels compared with sham stimulation. In addition, electrical DMN stimulation increased the serum levels of the anti‐inflammatory cytokine IL‐10 (p = 0.0284). In mice with CLP, electrical DMN stimulation resulted in significantly lower serum TNF levels compared with sham stimulation (p = 0.0291). These results indicate for the first time that electrical DMN stimulation alters circulating pro‐inflammatory and anti‐inflammatory levels in murine endotoxemia and sepsis. These findings are of interest for further development of bioelectronic approaches targeting DMN for therapeutic benefit in conditions, characterised by immune dysregulation and inflammation.

Sequencing of RNAs involved in sepsis-induced myocardial injury: role of Local sympathetic denervation

Sepsis has been a serious public health concern for a long time and is characterized by disrupted development of specific diagnostic criteria and novel therapies for sepsis is critically dependent on an understanding of the basic mechanisms immune homeostasis accompanied by infection and multiple organ dysfunction as determined assessed by the Sequential Organ Failure. Adult (4 to 6 months) male C57BL/6 mice, animal set in three groups, control group, Cecal Ligation and Puncture (CLP) group, CLP+ Local sympathetic denervation group 8 mice in each group. CLP model for created sepsis and local sympathetic denervation procedure for alleviated the effects of sepsis. Assessment of cardiac function by Millar Instruments, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and RNA extraction and sequencing. Our findings showed that the myocardial injury score and TNF-α level in both tissue and serum were significantly increased in the CLP group. CLP with local sympathetic denervation group mice had lower scores of myocardial injuries. Sepsis augments MCP-1 and KC level in myocardium. After 48 hours after CLP, the myocardial injury score. CLP with local sympathetic denervation alleviates sepsis-induced myocardial injury by inhibiting the NF-κB signaling pathway. Moreover, compared with the Ctrl group, the CLP with local sympathetic denervation group obtained fewer ncRNAs and mRNAs. Inconclusions, the NF-κB signaling pathway is associated with sepsis-induced myocardial injury, and local sympathetic denervation can relieve sepsis-induced myocardial injury by regulating the ncRNAs and mRNAs.