Cecal Ligation And Puncture Sepsis Research Articles

Tamibarotene targets heparin-binding protein for attenuating lung injury in sepsis.

Excessively active pulmonary inflammation is a hallmark of sepsis-induced lung damage. A synthetic retinoid drug called tamibarotene reduces inflammation in a variety of conditions, including acute promyelocytic leukemia (APL), renal fibrosis, and neuroinflammation. Its effect on sepsis-related lung injury, however, has not been explained. The purpose of the study was to investigate how tamibarotene affected lung damage induced by cecal ligation and puncture (CLP) procedure. A CLP sepsis mouse model was developed, and tamibarotene was pretreated to determine whether it improved lung injury and survival. The degree of lung injury was evaluated using the Hematoxylin and eosin staining and lung injury score. In order to determine pulmonary vascular permeability, measurements were taken for total protein and cell content of bronchoalveolar lavage fluid (BALF), wet/dry ratio of the lung, and Evans blue stain. The BALF inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and IL-17A were discovered by enzyme-linked immunosorbent serologic assay (ELISA). Then, the levels of heparin-binding protein (HBP), and phospho-nuclear factor kappa-B (p-NF-κB) P65, and NF-κB P65 were determined using ELISA and Western blot analysis, respectively. Tamibarotene considerably increases survival and lessens lung damage stimulated by sepsis. Specifically, tamibarotene significantly relieves pulmonary vascular permeability and inhibits inflammation response in sepsis. Moreover, we further confirmed that these ameliorating effects of tamibarotene on sepsis may be exerted by targeting HBP and regulating the activation of NF-κB signaling pathway. These findings demonstrated that tamibarotene lessens sepsis-induced lung injury, and the effect could be exerted by targeting HBP and thereby deregulating the NF-κB signaling pathway.

Less Severe Polymicrobial Sepsis in Conditional mgmt-Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis.

The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA suicide repair enzyme that might be important during sepsis but has never been explored. Then, the proteomic analysis of lipopolysaccharide (LPS)-stimulated wild-type (WT) macrophages increased proteasome proteins and reduced oxidative phosphorylation proteins compared with control, possibly related to cell injury. With LPS stimulation, mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated less profound inflammation; supernatant cytokines (TNF-α, IL-6, and IL-10) and pro-inflammatory genes (iNOS and IL-1β), with higher DNA break (phosphohistone H2AX) and cell-free DNA, but not malondialdehyde (the oxidative stress), compared with the littermate control (mgmtflox/flox; LysM-Cre-/-). In parallel, mgmt null mice (MGMT loss only in the myeloid cells) demonstrated less severe sepsis in the cecal ligation and puncture (CLP) model (with antibiotics), as indicated by survival and other parameters compared with sepsis in the littermate control. The mgmt null protective effect was lost in CLP mice without antibiotics, highlighting the importance of microbial control during sepsis immune modulation. However, an MGMT inhibitor in CLP with antibiotics in WT mice attenuated serum cytokines but not mortality, requiring further studies. In conclusion, an absence of mgmt in macrophages resulted in less severe CLP sepsis, implying a possible influence of guanine DNA methylation and repair in macrophages during sepsis.

Less Severe Sepsis in Cecal Ligation and Puncture Models with and without Lipopolysaccharide in Mice with Conditional Ezh2-Deleted Macrophages (LysM-Cre System).

Despite a previous report on less inflammatory responses in mice with an absence of the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, using a lipopolysaccharide (LPS) injection model, proteomic analysis and cecal ligation and puncture (CLP), a sepsis model that more resembles human conditions was devised. As such, analysis of cellular and secreted protein (proteome and secretome) after a single LPS activation and LPS tolerance in macrophages from Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) mice (Ezh2 null) and the littermate control mice (Ezh2fl/fl; LysM-Cre-/-) (Ezh2 control) compared with the unstimulated cells from each group indicated fewer activities in Ezh2 null macrophages, especially by the volcano plot analysis. Indeed, supernatant IL-1β and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1β and iNOS), TNF-α, and NF-κB (a transcription factor) were lower in Ezh2 null macrophages compared with the control. In LPS tolerance, downregulated NF-κB compared with the control was also demonstrated in Ezh2 null cells. In CLP sepsis mice, those with CLP alone and CLP at 2 days after twice receiving LPS injection, representing sepsis and sepsis after endotoxemia, respectively, symptoms were less severe in Ezh2 null mice, as indicated by survival analysis and other biomarkers. However, the Ezh2 inhibitor improved survival only in CLP, but not LPS with CLP. In conclusion, an absence of Ezh2 in macrophages resulted in less severe sepsis, and the use of an Ezh2 inhibitor might be beneficial in sepsis.

Abstract 395: Interferon-induced Transmembrane Protein 3 (ifitm3) Mediates Platelet Functions During Inflammatory Stress

Background: Thrombosis, precipitated by dysregulated platelet responses, contributes to adverse clinical outcomes, including organ failure and death during systemic inflammation (e.g., sepsis). Previously, our lab identified interferon-induced transmembrane protein 3 (IFITM3) as a mediator of anti-viral responses in platelets. However, whether IFITM3 mediates platelet functions and subsequent thrombosis during systemic inflammation is unknown. Methods: Cecal-ligation and Puncture (CLP) model of sepsis and Interferon alpha (IFNα)-induced sterile inflammation model was performed on WT and platelet- and MK-specific IFITM3 knockout strain (IFITM3 fl/fl PF4-Cre +/- ). P-Selectin and CD63 expression, Platelet Factor 4 (PF4) release, platelet-leukocyte aggregates, plasma cytokines (TNF-α, IL-6, CXCL1), as well as survival outcomes, platelet deposition and aggregates within organs (e.g. lungs), plasma aspartate aminotransferase, lactate dehydrogenase, D-Dimer levels were measured by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and immunohistochemistry. In vivo collagen-epinephrine-induced pulmonary embolism model, and ex vivo thrombus formation under flow using microfluidic devices were performed. Results: CLP sepsis or IFNα-induced sterile inflammation upregulated IFITM3 in platelets which were associated with significantly enhanced secretion of P-Selectin, PF4, and CD63, and increased mortality in a collagen-epinephrine-induced murine pulmonary embolism model. Biochemical assays demonstrated IFITM3 interacts with VAMP-8, the primary v-SNARE that drives platelet secretion. Platelets lacking IFITM3 showed reduced granule secretion and platelet-leukocyte aggregate formation. Sepsis-induced mortality, platelet-leukocyte aggregates, platelet deposition within lungs, and markers of organ failure and thrombosis (e.g., aspartate aminotransferase, lactate dehydrogenase, D-Dimer), and thrombus formation under flow were all significantly reduced in the absence of IFITM3 in CLP sepsis. Conclusions: Collectively, our findings suggest that IFITM3 is a novel regulator of secretion and aggregation in platelets and contributes to thrombotic events during systemic inflammation.

Early markers of Sepsis Cardiomyopathy in Murine Models by Echocardiography.

Strain echocardiography (SE) is a procedure for analyzing myocardial dysfunction that is known to be less dependent on preload and afterload of heart function. Unlike dimension-based parameters, like ejection fraction (EF) and fractional shortening (FS), SE measures cardiac function by tracking cardiac tissue deformation and anomalies throughout the cardiac cycle. Although SE is proven to locate myocardial ailments in various heart diseases, few studies exist regarding using SE relevant to sepsis pathophysiology. The study aimed to calculate the myocardial strain and strain rates, like longitudianl strain (LS), global radial strain (GRS), and global longitudinal strain (GLS), and to show these to be reduced earlier in cecal ligation and puncture (CLP) and lipopolysaccharide (LPS)-induced sepsis in coordination with an elevation of pro-inflammatory cytokines Methodology: Wild-type mice C57BL/6J (WT) were taken in this study and classified as CLP, LPS, and control groups. CLP surgery and LPS injection were given to induce sepsis. Endotoxemic septic shock was induced by intraperitoneal (IP) injection of LPS Escherichia coli. Echocardiography short axis views (SAX), longitudinal strain (LS), global circumferential strain (GCS), and global radial strain (GRS) were measured from the anterior and posterior positions of the septal and lateral walls of the heart. Real-time polymerase chain reaction (RT-PCR) was performed to evaluate post-CLP and LPS to analyze cardiac pro-inflammatory cytokines expressions. Inter- and intra-observer variables were tested by Bland-Altman analyses (BA). All data analysis was performed by GraphPad Prism 6 software. p<0.05 was taken as statistically significant. After 48 hours following CLP and LPS-induced sepsis, a significant declination in both longitudinal strain and strain rate (LS and LSR) was identified in the CLP and LPS groups compared to the control group. Strain depression in sepsis was linked with the up-regulation of pro-inflammatory cytokines in RT-PCR analysis. In the present study, we found myocardial strain and strain rate parameters, like LS, GRS, and GLS, to be reduced after CLP and LPS-induced sepsis in coordination with the elevation of pro-inflammatory cytokines.

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.

A Comparison Between 1 Day versus 7 Days of Sepsis in Mice with the Experiments on LPS-Activated Macrophages Support the Use of Intravenous Immunoglobulin for Sepsis Attenuation.

BackgroundBecause survival and death after sepsis are partly due to a proper immune adaptation and immune dysregulation, respectively, survivors and moribund mice after cecal ligation and puncture (CLP) sepsis surgery and in vitro macrophage experiments were explored.MethodsCharacteristics of mice at 1-day and 7-days post-CLP, the representative of moribund mice (an innate immune hyper-responsiveness) and survivors (a successful control on innate immunity), respectively. In parallel, soluble heat aggregated immunoglobulin (sHA-Ig), a representative of immune complex, was tested in lipopolysaccharide (LPS)-activated macrophages together with a test of intravenous immunoglobulin (IVIG), a molecule of adaptive immunity, on CLP sepsis mice.ResultsExcept for a slight increase in alanine transaminase (liver injury), IL-10, endotoxemia, and gut leakage (FITC-dextran assay), most of the parameters in survivors (7-days post-CLP) were normalized, with enhanced adaptive immunity, including serum immunoglobulin (using serum protein electrophoresis) and activated immune cells in spleens (flow cytometry analysis). The addition of sHA-Ig in LPS-activated macrophages reduced supernatant cytokines, cell energy (extracellular flux analysis), reactive oxygen species (ROS), several cell activities (proteomic analysis), and Fc gamma receptors (FcgRs) expression. The loss of anti-inflammatory effect of sHA-Ig in LPS-activated macrophages from mice with a deficiency on Fc gamma receptor IIb (FcgRIIb-/-), the only inhibitory signaling of FcgRs family, when compared with wild-type macrophages, implying the FcgRIIb-dependent mechanism. Moreover, IVIG attenuated sepsis severity in CLP mice as evaluated by serum creatinine, liver enzyme (alanine transaminase), serum cytokines, spleen apoptosis, and abundance of dendritic cells in the spleen (24-h post-CLP) and survival analysis.ConclusionImmunoglobulin attenuated LPS-activated macrophages, partly, through the reduced cell energy of macrophages and might play a role in sepsis immune hyper-responsiveness. Despite the debate over IVIG’s use in sepsis, IVIG might be beneficial in sepsis with certain conditions.

Dabigatran activates inflammation resolution by promoting fibrinogen-like protein 2 shedding and RvD5n-3 DPA production.

Rationale: The interaction between coagulation and inflammation resolution remains elusive. We recently highlighted a link between fibrinogen-like protein 2 (Fgl2) and a specialized pro-resolving mediator (SPM)-n-3 docosapentaenoic acid-derived resolvin D5 (RvD5n-3 DPA) in sepsis. This study aimed to investigate the functions of commonly used anticoagulants warfarin, dabigatran and heparin in regulating inflammation resolution.Methods: Peripheral blood was collected from clinical sepsis patients and healthy control for the determination of indicated indexes. Mouse sepsis models of zymosan-induced peritonitis and cecal ligation and puncture (CLP) were employed for the measurement of inflammation- and coagulation-related indexes. Western-blotting, ELISA and flow cytometry were applied to assess proteins. UPLC-MS/MS was used to evaluate lipid metabolites.Results: Here we report that the transmembrane Fgl2 (mFgl2) was positively associated with coagulation, while soluble Fgl2 (sFgl2) level correlated with the enhanced number of peripheral blood mononuclear cells in the sepsis patients. The anticoagulants dabigatran and warfarin attenuated zymosan-induced peritonitis, which was not shared by heparin, while only dabigatran significantly improved sepsis survival in the CLP sepsis mouse model. Although these anticoagulants consistently inhibited pro-inflammatory mediators including prostaglandin E2 and leukotriene B4, only dabigatran increased sFgl2 at both the initiation and resolution phases of inflammation. Mechanistically, dabigatran elicited the shedding of sFgl2 via prothrombin-related metalloproteases, thereby enhanced the subsequent biosynthesis of RvD5n-3 DPA via STAT6-ALOX15 axis. Blocking metalloproteases or ALOX15 significantly impaired dabigatran-enhanced macrophage efferocytosis in vitro, as well as delayed the dabigatran-accelerated inflammation resolution in vivo.Conclusions: Our findings identify the dual anti-inflammatory and pro-resolving actions of dabigatran, through promoting sFgl2-triggered RvD5n-3 DPA production, which has important implications for promoting tissue homeostasis of sepsis.

CD4 and CD8 T Cell Memory Interactions Alter Innate Immunity and Organ Injury in the CLP Sepsis Model.

The role of T cell memory in sepsis is poorly understood. Recent work has demonstrated that mice exposed to frequent antigenic stimulation, in contrast to laboratory mice, better recapitulate the human T cell repertoire. This difference may profoundly alter responses to inflammatory insults. We induced isolated T cell memory by inoculating C57Bl/6 mice with an anti-CD3ϵ activating antibody, a process we term “immune education.” These mice were subjected to the cecal ligation and puncture (CLP) model of sepsis and responses were compared to those of isotype-treated controls. CLP-induced increases in 1) CD4 T cell production and serum levels of IFNγ, 2) CD8 T cell granzyme B levels, and 3) innate cell function were all more pronounced in educated mice than in control mice. Immune education increased CLP-induced liver injury and decreased survival. The differences in responses to CLP were not recapitulated in mice with either isolated CD4 or isolated CD8 T cell memory. Relative to controls, CLP in educated CD8−/− mice (isolated CD4 memory) increased monocyte-derived dendritic cells. Combined CD4 and CD8 memory did not increase monocyte-derived dendritic cells; this combination recapitulated increases in neutrophil and inflammatory monocyte numbers in educated wild-type mice. Induction of T cell memory prior to CLP alters immune responses, organ function, and survival. Both CD4 and CD8 memory T cells play important and independent roles in this response. These findings have profound implications for the development of murine models of human inflammatory disorders such as infection and sepsis.

Endothelial HSPA12B Exerts Protection Against Sepsis-Induced Severe Cardiomyopathy via Suppression of Adhesion Molecule Expression by miR-126.

Heat shock protein A12B (HSPA12B) is predominately expressed in endothelial cells (ECs) and has been reported to protect against cardiac dysfunction from endotoxemia or myocardial infarction. This study investigated the mechanisms by which endothelial HSPA12B protects polymicrobial sepsis–induced cardiomyopathy. Wild-type (WT) and endothelial HSPA12B knockout (HSPA12B–/–) mice were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP). Cecal ligation and puncture sepsis accelerated mortality and caused severe cardiac dysfunction in HSPA12B–/– mice compared with WT septic mice. The levels of adhesion molecules and the infiltrated immune cells in the myocardium of HSPA12B–/– septic mice were markedly greater than in WT septic mice. The levels of microRNA-126 (miR-126), which targets adhesion molecules, in serum exosomes from HSPA12B–/– septic mice were significantly lower than in WT septic mice. Transfection of ECs with adenovirus expressing HSPA12B significantly increased miR-126 levels. Increased miR-126 levels in ECs prevented LPS-stimulated expression of adhesion molecules. In vivo delivery of miR-126 carried by exosomes into the myocardium of HSPA12B–/– mice significantly attenuated CLP sepsis increased levels of adhesion molecules, and improved CLP sepsis–induced cardiac dysfunction. The data suggest that HSPA12B protects against sepsis-induced severe cardiomyopathy via regulating miR-126 expression which targets adhesion molecules, thus decreasing the accumulation of immune cells in the myocardium.

Presepsin Levels in Experimental Sepsis and Massive Bowel Resection Models in Rats.

Presepsin is a useful biomarker for diagnosing sepsis. This study aimed to investigate the relationship between oxidative stress and presepsin levels in animal models. Sprague-Dawley rats were used for cecal ligation and puncture (CLP) and to generate massive bowel resection (MBR) models. Trunk blood was collected for analysis of presepsin. Liver and intestinal tissue samples were taken to determine oxidative stress parameters. Presepsin levels in MBR and CLP sepsis models were higher than those in control groups. Reactive oxygen and nitrogen species (RONS) and malondialdehyde levels were increased in the liver and small intestine of rats in both models, whereas glutathione levels were decreased. Presepsin levels and RONS may be released by the same mechanism which is closely associated with the progression of sepsis and inflammation in both CLP and MBR models.

Lactate up-regulates the expression of PD-L1 in kidney and causes immunosuppression in septic Acute Renal Injury

This study aims to explore the mechanism of immunosuppression in septic Acute Renal Injury (AKI) and the role of programmed death-1 (PD-1/PD-L1) pathway in septic AKI. This study established a septic AKI model by Cecal ligation and puncture (CLP) in C57/B6 mice, ELISA was used to test the level of lactate and creatinine in serum, blood was collected for flow cytometry and kidney samples for Western blot analyses. This study further analyzed the expression of PD-L1 in kidney and the expression of PD-1 in CD4+, CD8+ T cell, and the number of CD3+ T cells to identify apoptosis in T cells in the blood. The CLP sepsis model induced AKI in C57/B6 mice; The expression of PD-1 and PD-L1 were increased in septic AKI mice; PD-1/PD-L1 induced apoptosis in T cells: the number of lymphocytes decreased by 64%, while the number of CD3+ T cells decreased by 27% compared with the sham group; Results also indicated that lactate up-regulates expression of PD-L1 in the kidney. Lactate activated PD-1/PD-L1 pathway can induce immunosuppression by inducing apoptosis in lymphocytes in septic AKI. Moreover, blocking the receptor of lactate or PD-1/PD-L1 might be a new therapy for septic AKI.

Body temperature and mouse scoring systems as surrogate markers of death in cecal ligation and puncture sepsis

BackgroundDespite increasing ethical standards for conducting animal research, death is still often used as an endpoint in mouse sepsis studies. Recently, the Murine Sepsis Score (MSS), Mouse Clinical Assessment Score for Sepsis (M-CASS), and Mouse Grimace Scale (MGS) were developed as surrogate endpoint scoring systems for assessing pain and disease severity in mice. The objective of our study was to compare the effectiveness of these scoring systems and monitoring of body temperature for predicting disease progression and death in the cecal ligation and puncture (CLP) sepsis model, in order to better inform selection of surrogate endpoints for death in experimental sepsis.MethodsC57Bl/6J mice were subjected to control sham surgery, or moderate or severe CLP sepsis. All mice were monitored every 4 h for surrogate markers of death using modified versions of the MSS, M-CASS, and MGS scoring systems until 24 h post-operatively, or until endpoint (inability to ambulate) and consequent euthanasia.ResultsThirty percent of mice subjected to moderate severity CLP reached endpoint by 24 h post-CLP, whereas 100% undergoing severe CLP reached endpoint within 20 h. Modified MSS, M-CASS, and MGS scores all increased, while body temperature decreased, in a time-dependent and sepsis severity-dependent manner, although modified M-CASS scores showed substantial variability. Receiver operating characteristic curves demonstrate that the last recorded body temperature (AUC = 0.88; 95% CI 0.77–0.99), change in body temperature (AUC = 0.89; 95% CI 0.78–0.99), modified M-CASS (AUC = 0.93; 95% CI 0.85–1.00), and modified MSS (AUC = 0.95; 95% CI 0.88–1.01) scores are all robust for predicting death in CLP sepsis, whereas modified MGS (AUC = 0.78; 95% CI 0.63–0.92) is less robust.ConclusionsThe modified MSS and body temperature are effective markers for assessing disease severity and predicting death in the CLP model, and should thus be considered as valid surrogate markers to replace death as an endpoint in mouse CLP sepsis studies.

Interleukin-33 contributes to ILC2 activation and early inflammation-associated lung injury during abdominal sepsis.

Sepsis is defined as infection with organ dysfunction due to a dysregulated immune response. The lung is one of the most vulnerable organs at the onset of sepsis. Interleukin (IL)-33 can be released by injured epithelial and endothelial cells in the lung and regulate immune activation and infiltration. Therefore, we postulated that IL-33 would contribute to the immune response in the lung during sepsis. Using the cecal ligation and puncture (CLP) sepsis model, we show here that IL-33 contributes significantly to both sepsis-induced inflammation in the lung and systemic inflammatory response in the early phase of sepsis. Despite the higher intra-peritoneal bacterial burden, the absence of IL-33 resulted in less infiltration of neutrophils and monocytes into the lungs in association with lower circulating, lung and liver cytokine levels as well as reduced lung injury at 6h after sepsis. IL-33 was required for the upregulation of IL-5 in type 2 Innate Lymphoid Cells (ILC2), while IL-5 neutralization suppressed neutrophil and monocyte infiltration in the lungs during CLP sepsis. This reduction in leukocyte infiltration in IL-33-deficient mice was reversed by administration of recombinant IL-5. These results indicate that IL-33 plays a major role in the local inflammatory changes in the lung, in part, by regulating IL-5 and this axis contributes to lung injury early after the onset of sepsis.

Dose-related effects of dexmedetomidine on immunomodulation and mortality to septic shock in rats.

Dexmedetomidine has already been used in septic patients as a new sedative agent, few studies have examined its effects on immunomodulation. Therefore, the authors have designed a controlled experimental study to characterize the immunomodulation effects of dexmedetomidine in the cecal ligation and puncture (CLP) model in rats. After CLP, 48 Wistar rats were randomly allocated into four groups: (1) CLP group; (2) small-dose treatment group (2.5 μg·kg-1·h-1); (3) medium-dose treatment group (5.0 μg·kg-1· h-1); and (4) large-dose treatment group (10.0 μg·kg-1·h-1). HLA-DR and plasma cytokine (IL-4, IL-6, IL-10 and TNF-α) levels were measured, and the mean arterial blood pressure (MAP), heart rate (HR), arterial blood gases, lactate concentrations and mortality were also documented. The HLA-DR level, inflammatory mediator levels, MAP and HR had no obvious changes among Dexmedetomidine treatment groups (DEX groups). Compared with the CLP group, the DEX groups exhibited decreased HLA-DR levels (Pgroup=0.0202) and increased IL-6 production, which was increased at 3 h (P= 0.0113) and was then attenuated at 5 h; additionally, the DEX groups exhibited decreased HR (P<0.001) while maintaining MAP (Pgroup=0.1238), and remarkably improving lactate (P<0.0001). All of these factors led to a significant decrease in the mortality, with observed rates of 91.7%, 66.7%, 25% and 18% for the CLP, DEX2.5, DEX5.0, DEX10.0 groups, respectively. Dexmedetomidine treatment in the setting of a CLP sepsis rat model has partially induced immunomodulation that was initiated within 5 h, causing a decreased HR while maintaining MAP, remarkably improving metabolic acidosis and improving mortality dose-dependently.

A comparative study of changes of autophagy in rat models of CLP versus LPS induced sepsis.

In the present study, two different rat models of sepsis, cecal ligation and puncture (CLP), and lipopolysaccharide (LPS), were established. Changes in autophagy in both models were compared using transmission electron microscopy (TEM), immunohistochemistry, western blotting, and quantitative polymerase chain reaction techniques. Consequently, TEM analysis revealed autophagic bodies in the CLP and LPS sepsis models. In addition, autophagy-related protein LC3 A-specific staining was detected in the cytoplasm. However, analysis of protein and gene expression levels revealed a statistically significant increase in autophagic activity 12 and 24 h following induction of the CLP group, and 2 h following induction of the LPS group. Thus, it was concluded that both models of sepsis exhibited increased autophagic activity of the cardiomyocytes over time. The LPS model was superior to the CLP model in perturbation of molecular biological mechanisms, while the latter would be more likely suited for the study of physiological functions.

Enhanced Glycolytic Metabolism Contributes to Cardiac Dysfunction in Polymicrobial Sepsis.

Cardiac dysfunction is present in >40% of sepsis patients and is associated with mortality rates of up to 70%. Recent evidence suggests that glycolytic metabolism plays a critical role in host defense and inflammation. Activation of Toll-like receptors on immune cells can enhance glycolytic metabolism. This study investigated whether modulation of glycolysis by inhibition of hexokinase will be beneficial to septic cardiomyopathy. Male C57B6/J mice were treated with a hexokinase inhibitor (2-deoxy-d-glucose [2-DG], 0.25-2 g/kg, n = 6-8) before cecal ligation and puncture (CLP) induced sepsis. Untreated septic mice served as control. Sham surgically operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate. Sepsis-induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction and fractional shortening in 2-DG-treated septic mice were significantly (P < .05) greater than in untreated CLP mice. 2-DG administration also significantly improved survival outcome, reduced kidney and liver injury, attenuated sepsis-increased serum levels of tumor necrosis factor α and interleukin 1β as well as lactate, and enhanced the expression of Sirt1 and Sirt3 in the myocardium, which play an important role in mitochondrial function and metabolism. In addition, 2-DG administration suppresses sepsis-increased expression of apoptotic inducers Bak and Bax as well as JNK phosphorylation in the myocardium. Glycolytic metabolism plays an important role in mediating sepsis-induced septic cardiomyopathy. The mechanisms may involve regulation of inflammatory response and apoptotic signaling.

Gastrointestinal Leakage Detected by Serum (1→3)-β-D-Glucan in Mouse Models and a Pilot Study in Patients with Sepsis.

Gastrointestinal (GI) leakage is believed to exacerbate sepsis and new, validated markers of GI barrier performance might benefit clinical decision-making. Serum (1→3)-β-D-glucan (BG) was evaluated as a potential GI leakage marker. Serum BG was tested in several mouse models of GI leakage, including dextran sulfate solution (DSS) administration, endotoxin (LPS) injection, and cecal ligation and puncture sepsis (CLP). Serum BG titer was also evaluated in patients with sepsis and septic shock, for comparison.With 0.75% DSS administration, BG increased only after oral administration of heat-killed C. albicans, but increased spontaneously with 1.5% DSS. In the LPS and CLP models, BG increased as early as 1 h and at 12 h after LPS administration and surgery, respectively. GI leakage was confirmed by orthogonal validation methods including FITC-dextran oral administration in the DSS, LPS, and CLP models and, in the DSS model, with urine sucralose after oral administration and serum endotoxemia. IL-6 increased in parallel with serum BG. Serum BG or IL-6, at 18 h, anticipated sepsis mortality in the CLP model.Analysis of serum BG from patients with febrile neutropenic sepsis (N = 49) and febrile non-neutropenic sepsis (N = 39) demonstrated BG elevation. Patients with bacterial septic shock had serum BG titers similar to levels observed in invasive fungal disease, regardless of febrile neutropenia. Serum BG was lower in less severe cases of bacterial sepsis. Elevated serum IL-6 was associated with GI leakage and elevated serum BG.Serum BG may have potential as a sepsis/septic shock biomarker and further study in this context is warranted.

Protective effect of recombinant Trichinella 53-kDa protein in sepsis and the effect on macrophages.

The survival rate of the recombinant Trichinella 53-kDa protein infected by the cecal ligation and puncture (CLP) model of sepsis in rats and the expression difference of macrophages were analyzed. Eighteen clean SD rats were selected for the present study. The rats were divided into the sham operation (n=5), control (n=5) and experimental (n=8) groups. The rats in the sham operation group underwent cecum division and suture with routine therapy for cure. The rats in the control and experimental groups were placed in the CLP model of sepsis in rats. The experimental group was administered recombinant Trichinella 53-kDa protein in advance, and the control group was administered the same dose of placebo. The survival rate of the rats within 6 and 12 h, the macrophage expression ratio, and the differences of the expression levels Th1-type cytokines IFN-γ and tumor necrosis factor (TNF)-α, and the Th2 type cytokines interleukin-4 (IL-4) and IL-13 were analyzed. The survival rate of rats in the experimental group was higher than that of the control group with a statistically significant difference (P<0.05). The expression ratio of the macrophages received from the different handling methods of the rats in the experimental group was higher than that of the control group. The difference was statistically significant (P<0.05). The expression levels of the Th1-type cytokines IFN-γ and TNF-α of the rats in the experimental group was higher than that of the control group, while the expression level of the Th2-type cytokines IL-4 and IL-13 was higher than that of the control group. The difference was statistically significant (P<0.05). In conclusion, recombinant Trichinella 53-kDa protein can increase the survival rate following infection with CLP sepsis, which may be associated with the improvement of the macrophages and the adjustment of the expression of Th2 cytokines.

Factor XI Deficiency Alters the Cytokine Response and Activation of Contact Proteases during Polymicrobial Sepsis in Mice.

Sepsis, a systemic inflammatory response to infection, is often accompanied by abnormalities of blood coagulation. Prior work with a mouse model of sepsis induced by cecal ligation and puncture (CLP) suggested that the protease factor XIa contributed to disseminated intravascular coagulation (DIC) and to the cytokine response during sepsis. We investigated the importance of factor XI to cytokine and coagulation responses during the first 24 hours after CLP. Compared to wild type littermates, factor XI-deficient (FXI-/-) mice had a survival advantage after CLP, with smaller increases in plasma levels of TNF-α and IL-10 and delayed IL-1β and IL-6 responses. Plasma levels of serum amyloid P, an acute phase protein, were increased in wild type mice 24 hours post-CLP, but not in FXI-/- mice, supporting the impression of a reduced inflammatory response in the absence of factor XI. Surprisingly, there was little evidence of DIC in mice of either genotype. Plasma levels of the contact factors factor XII and prekallikrein were reduced in WT mice after CLP, consistent with induction of contact activation. However, factor XII and PK levels were not reduced in FXI-/- animals, indicating factor XI deficiency blunted contact activation. Intravenous infusion of polyphosphate into WT mice also induced changes in factor XII, but had much less effect in FXI deficient mice. In vitro analysis revealed that factor XIa activates factor XII, and that this reaction is enhanced by polyanions such polyphosphate and nucleic acids. These data suggest that factor XI deficiency confers a survival advantage in the CLP sepsis model by altering the cytokine response to infection and blunting activation of the contact (kallikrein-kinin) system. The findings support the hypothesis that factor XI functions as a bidirectional interface between contact activation and thrombin generation, allowing the two processes to influence each other.