Animal Models Of Sepsis Research Articles

Identifying Therapeutic Targets for Sepsis Research: A Characterization Study of the Inflammatory Players in the Cecal Ligation and Puncture Model.

During sepsis, disturbed gastrointestinal motility and increased mucosal permeability can aggravate sepsis due to the increased risk of bacterial translocation. To help identify new therapeutic targets, there is a need for animal models that mimic the immunological changes in the gastrointestinal tract as observed during human sepsis. We therefore characterized in detail the gastrointestinal neuroimmune environment in the cecal ligation and puncture (CLP) model, which is the gold standard animal model of microbial sepsis. Mice were sacrificed at day 2 and day 7, during which gastrointestinal motility was assessed and cytokines were measured in the serum and the colon. In the spleen, lymph nodes, ileum, and colon, subsets of leukocyte populations were identified by flow cytometry. Septic animals displayed an impaired gastrointestinal motility at day 2 and day 7. Two days post-CLP, increased serum and colonic levels of proinflammatory cytokines were measured. Flow cytometry revealed an influx of neutrophils in the colon and ileum, increased numbers of macrophages in the spleen and mesenteric lymph nodes, and an enhanced number of mast cells in all tissues. At day 7 post-CLP, lymphocyte depletion was observed in all tissues coinciding with increased IL-10 and TGF-β levels, as well as increased colonic levels of IL-17A and IFN-γ. Thus, CLP-induced sepsis in mice results in simultaneous activation of pro- and anti-inflammatory players at day 2 and day 7 in different tissues, mimicking human sepsis.

Vascular Effects of Adrenomedullin and the Anti-Adrenomedullin Antibody Adrecizumab in Sepsis.

Sepsis remains a major scientific and medical challenge, for which, apart from significant refinements in supportive therapy, treatment has barely changed over the last few decades. During sepsis, both vascular tone and vascular integrity are compromised, and contribute to the development of shock. The free circulating peptide adrenomedullin (ADM) is involved in the regulation of the endothelial barrier function and tone of blood vessels. Several animal studies have shown that ADM administration improves outcome of sepsis. However, in higher dosages, ADM administration may cause hypotension, limiting its clinical applicability. Moreover, ADM has a very short half-life and easily adheres to surfaces, further hampering its clinical use. The non-neutralizing anti-ADM antibody Adrecizumab (HAM8101) which causes a long-lasting increase of plasma ADM has shown promising results in animal models of systemic inflammation and sepsis; it reduced inflammation, attenuated vascular leakage, and improved hemodynamics, kidney function, and survival. Combined with an excellent safety profile derived from animal and phase I human studies, Adrecizumab represents a promising candidate drug for the adjunctive treatment of sepsis. In this review, we first provide a brief overview of the currently available data on the role of adrenomedullin in sepsis and describe its effects on endothelial barrier function and vasodilation. Furthermore, we provide a novel hypothesis concerning the mechanisms of action through which Adrecizumab may exert its beneficial effects in sepsis.

A systematic investigation on animal models of cyclosporine A combined with Escherichia coli to simulate the immunosuppressive status of sepsis patients before onset

Immunosuppression is an important mechanism for the development of sepsis pathology, and is the key to the high mortality of sepsis. However, patients appear to be immunocompromised before sepsis onset due to lack of enough attention. Present sepsis models cannot fully mimic the onset of sepsis in patients. Hence, effective treatments in animal experiments could not be transformed into clinical application. In the present study, we improved the animal model of sepsis and used cyclosporine A immunosuppressive mice to make it closer to immune status before the onset of sepsis, followed by the intraperitoneal injection of Escherichia coli (E. coli) CMCC (B) 44,102 standard strain to produce the immunocompromised sepsis model. This trial systematically evaluates the new immunosuppressive sepsis model. Compared with routine sepsis models, the release of inflammatory factors in the new sepsis model was insufficient, blood bacteria were more cultured, diffuse intravascular coagulation (DIC) was more severe, lung, liver and kidney damage were heavier, and mortality rate was higher. In conclusion, the new sepsis model can mimic the patient's pre-onset immunocompromised state, is suitable for the development and evaluation of new methods of sepsis, and solves the controversy of sepsis treatment, providing new ideas and direction.

Nucleic-acid based gene therapy approaches for sepsis

Despite advances in overall medical care, sepsis and its sequelae continue to be an embarrassing clinical entity with an unacceptably high mortality rate. The central reason for high morbidity and high mortality of sepsis and its sequelae is the lack of an effective treatment. Previous clinical trials have largely failed to identify an effective therapeutic target to improve clinical outcomes in sepsis. Thus, the key goal favoring the outcome of septic patients is to devise innovative and evolutionary therapeutic strategies. Gene therapy can be considered as one of the most promising novel therapeutic approaches for nasty disorders. Since a number of transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), play a pivotal role in the pathophysiology of sepsis that can be characterized by the induction of multiple genes and their products, sepsis may be regarded as a gene-related disorder and gene therapy may be considered a promising novel therapeutic approach for treatment of sepsis. In this review article, we provide an up-to-date summary of the gene-targeting approaches, which have been developed in animal models of sepsis. Our review sheds light on the molecular basis of sepsis pathology for the development of novel gene therapy approaches and leads to the conclusion that future research efforts may fully take into account gene therapy for the treatment of sepsis.

Plasma Adsorption Membranes Are Able to Efficiently Remove High Mobility Group Box-1 (HMGB-1).

High Mobility Group Box 1 (HMGB-1) is a 30 kDa protein that is a lethal mediator in sepsis and is a recognized therapeutic target. However, no consensus has been reached for acute blood purification therapy as a treatment for sepsis targeting HMGB-1. Previous studies demonstrated that a high anti-HMGB-1 antibody titer and the suppression of HMGB-1 activity improved survival rate in animal sepsis models. The aim of this study was to evaluate whether plasma adsorption therapy is able to decrease the level of HMGB-1, representing a new potential treatment strategy against sepsis. Plasma adsorption therapy has been known as a treatment for various autoimmune diseases. Three different adsorbent columns, including TR-350 (IM-TR), PH-350 (IM-PH), and BRS-350 (BRS), were used in this study for comparison. We made a 1/350 scale of these three columns. Fetal bovine serum (FBS) contains a significant amount of HMGB-1. After priming with saline, FBS was passed through the columns and the adsorption rates of HMGB-1 at 25 minutes, 50 minutes, and 75 minutes were evaluated. The total adsorbed HMGB-1 level at 75 minutes was also calculated. The highest adsorption rate and total adsorbed amount of HMGB-1 were observed in IM-TR, followed by BRS and IM-PH. IM-TR showed a decline in adsorption rate over time. BRS showed a steady adsorption rate at all time points. IM-TR removed HMGB-1 significantly more than IM-PH and BRS. Our findings showed that plasma adsorption therapy efficiently adsorbed HMGB-1 and could be safely applied for the treatment of sepsis.

Cellular Mechanisms of Myocardial Depression in Porcine Septic Shock.

The complex pathogenesis of sepsis and septic shock involves myocardial depression, the pathophysiology of which, however, remains unclear. In this study, cellular mechanisms of myocardial depression were addressed in a clinically relevant, large animal (porcine) model of sepsis and septic shock. Sepsis was induced by fecal peritonitis in eight anesthetized, mechanically ventilated, and instrumented pigs of both sexes and continued for 24 h. In eight control pigs, an identical experiment but without sepsis induction was performed. In vitro analysis of cardiac function included measurements of action potentials and contractions in the right ventricle trabeculae, measurements of sarcomeric contractions, calcium transients and calcium current in isolated cardiac myocytes, and analysis of mitochondrial respiration by ultrasensitive oxygraphy. Increased values of modified sequential organ failure assessment score and serum lactate levels documented the development of sepsis/septic shock, accompanied by hyperdynamic circulation with high heart rate, increased cardiac output, peripheral vasodilation, and decreased stroke volume. In septic trabeculae, action potential duration was shortened and contraction force reduced. In septic cardiac myocytes, sarcomeric contractions, calcium transients, and L-type calcium current were all suppressed. Similar relaxation trajectory of the intracellular calcium-cell length phase-plane diagram indicated unchanged calcium responsiveness of myofilaments. Mitochondrial respiration was diminished through inhibition of Complex II and Complex IV. Defective calcium handling with reduced calcium current and transients, together with inhibition of mitochondrial respiration, appears to represent the dominant cellular mechanisms of myocardial depression in porcine septic shock.

Mycobacterium tuberculosis PPE18 Protein Reduces Inflammation and Increases Survival in Animal Model of Sepsis.

Mycobacterium tuberculosis PPE18 is a member of the PPE family. Previous studies have shown that recombinant PPE18 (rPPE18) protein binds to TLR2 and triggers a signaling cascade which reduces levels of TNF-α and IL-12, and increases IL-10 in macrophages. Because TNF-α is a major mediator of the pathophysiology of sepsis and blocking inflammation is a possible line of therapy in such circumstances, we tested the efficacy of rPPE18 in reducing symptoms of sepsis in a mouse model of Escherichia coli-induced septic peritonitis. rPPE18 significantly decreased levels of serum TNF-α, IL-1β, IL-6, and IL-12 and reduced organ damage in mice injected i.p. with high doses of E. coli Peritoneal cells isolated from rPPE18-treated mice had characteristics of M2 macrophages which are protective in excessive inflammation. Additionally, rPPE18 inhibited disseminated intravascular coagulation, which can cause organ damage resulting in death. rPPE18 was able to reduce sepsis-induced mortality when given prophylactically or therapeutically. Additionally, in a mouse model of cecal ligation and puncture-induced sepsis, rPPE18 reduced TNF-α, alanine transaminase, and creatinine, attenuated organ damage, prevented depletion of monocytes and lymphocytes, and improved survival. Our studies show that rPPE18 has potent anti-inflammatory properties and can serve as a novel therapeutic to control sepsis.

Blockade of glutamate receptor ameliorates lipopolysaccharide-induced sepsis through regulation of neuropeptides.

Glutamate receptors (N-methyl-d-aspartate receptor (NMDAR)) are expressed mainly in the central nervous system (CNS), but several potentially important exceptions are worth mentioning. Recently, NMDAR, a glutamate receptor, has been reported to be found in the lungs. NMDAR is activated in acute lung injury (ALI). Here, the present experiment was designed to examine whether NMDAR blockade (MK-801) ameliorates ALI through affecting neuropeptides in LPS-induced sepsis animal models. Male Kunming mice were divided into control group, LPS group, control + MK-801 group, and LPS + MK-801 group. Bronchoalveolar lavage fluid (BALF) was collected and evaluated. The lung histological pathology was assayed by immunocytochemistry staining. Western blot was used to measure PGP9.5, substance P (SP), and vasoactive intestinal polypeptide (VIP). Results showed that LPS-induced mice animal models were ameliorated by co-treatment with the MK-801, an uncompetitive NMDAR antagonist. Moreover, the protective effects of MK-801 attributed to the increased secretion of VIP and decreased secretion of SP. The results of the present study indicated that the blockade of NMDAR may represent a promising therapeutic strategy for the treatment of sepsis-associated ALI through regulation of neuropeptides.

Nuclear PTEN enhances the maturation of a microRNA regulon to limit MyD88-dependent susceptibility to sepsis.

Sepsis-induced organ damage is caused by systemic inflammatory response syndrome (SIRS), which results in substantial comorbidities. Therefore, it is of medical importance to identify molecular brakes that can be exploited to dampen inflammation and prevent the development of SIRS. We investigated the role of phosphatase and tensin homolog (PTEN) in suppressing SIRS, increasing microbial clearance, and preventing lung damage. Septic patients and mice with sepsis exhibited increased PTEN expression in leukocytes. Myeloid-specific Pten deletion in an animal model of sepsis increased bacterial loads and cytokine production, which depended on enhanced myeloid differentiation primary response gene 88 (MyD88) abundance and resulted in mortality. PTEN-mediated induction of the microRNAs (miRNAs) miR125b and miR203b reduced the abundance of MyD88. Loss- and gain-of-function assays demonstrated that PTEN induced miRNA production by associating with and facilitating the nuclear localization of Drosha-Dgcr8, part of the miRNA-processing complex. Reconstitution of PTEN-deficient mouse embryonic fibroblasts with a mutant form of PTEN that does not localize to the nucleus resulted in retention of Drosha-Dgcr8 in the cytoplasm and impaired production of mature miRNAs. Thus, we identified a regulatory pathway involving nuclear PTEN-mediated miRNA generation that limits the production of MyD88 and thereby limits sepsis-associated mortality.

Hippocampal β2‑microglobulin mediates sepsis‑induced cognitive impairment.

Acute brain dysfunction is a frequent complication in sepsis patients and is associated with long‑term neurocognitive consequences and increased mortality, yet the underlying mechanism remains unclear. Emerging evidence has suggested that β2‑microglobulin [a component of major histocompatibility complex (MHC) classI molecules] is involved in cognitive dysfunction in various neurological diseases. Therefore, the present study tested the hypothesis that β2‑microglobulin in the brain also mediates sepsis‑induced cognitive impairment. In the present study, wild‑type and antigen processing 1 (Tap1)‑deficient mice (Tap1‑/‑) were subjected to cecal ligation and puncture (CLP). Survival rate, cognitive function, and biochemical analysis were performed at the indicated time points. The data revealed that CLP induced anxiety‑like behavior and impaired hippocampal‑dependent contextual memory in wild‑type mice, which was accompanied by hippocampal microglial activation, increased level of interleukin‑1β, and decreased concentrations of brain derived neurotrophic factor and postsynaptic density protein 95. Notably, it was demonstrated that Tap1‑/‑ mice with reduced cell surface expression of MHCI protected mice from anxiety‑like behavior and impaired hippocampal‑dependent contextual memory and reversed most of these biochemical parameters following sepsis development. In summary, the results of the present study suggest that β2‑microglobulin negatively regulates cognitive impairment in an animal model of sepsis induced by CLP.

Citrullinated Histone 3 Contributes to Endothelial Barrier Dysfunction During Sepsis

Circulating extracellular histones are known to contribute to tissue injury in systemic inflammatory responses to infection, trauma, and burns. The mechanisms of histone modifications and their effects on inflammation are currently being investigated. One such modification, namely citrullination of histone 3, is catalyzed by the peptidylarginine deiminase 4 (PAD4) enzyme and is thought to initiate the process of NETosis (the release of neutrophil extracellular traps) in response to infection. NETs are composed of a chromatin web with attached proteases and histones, including citrullinated histone 3 (citH3). This study aimed to identify the effects of citH3 on endothelial barrier dysfunction during inflammation. We identified increased citH3 in the tissues and plasma of mice subjected to cecal ligation and puncture, a clinically relevant animal model of polymicrobial sepsis. In vivo, stimulation of mesenteric microvessels with citH3 elicited plasma protein leakage into the surrounding tissue. In cultured endothelial cell monolayers, treatment with citH3 induced barrier dysfunction as indicated by reduced transendothelial electrical resistance. Endothelial monolayer dysfunction partially resulted from disrupted adherens junction proteins VE‐cadherin and β‐catenin, as well as disturbed actin cytoskeletal homeostasis, as demonstrated by immunofluorescence with confocal microscopy. Taken together, the data suggest citH3 induces endothelial barrier dysfunction, which can exacerbate the inflammatory response during sepsis.Support or Funding InformationThis work is supported by NIH R01s HL070752 and HL126646.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oral supplementation with ursolic acid ameliorates sepsis-induced acute kidney injury in a mouse model by inhibiting oxidative stress and inflammatory responses.

Ursolic acid (UA) as a multiple bioactive native compound has recently been demonstrated to treat sepsis in animal models. However, the beneficial effects of UA in sepsis-induced acute kidney injury (AKI) are not completely understood. In the present study, the effect of UA on sepsis-induced AKI in cecal ligation and puncture (CLP) surgery mice was investigated. Renal histomorphological analysis was performed by hematoxylin and eosin staining. The expression of inflammatory markers in the kidney of septic mice was measured by reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that UA administration improved survival in septic mice induced by CLP surgery. The treatment with UA revealed protection against AKI induced by CLP surgery, including the alleviation of glomerular damage and vacuolization in the proximal tubules. In addition, the effects of UA on oxidative stress and inflammation in septic mice were determined. The findings suggested that UA may protect against sepsis-induced AKI by inhibiting reactive oxygen species and inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β and IL-6, in the kidney from septic mice. Finally, UA inhibited CLP-induced activation of nuclear factor-κB signaling in the kidney from septic mice. The findings of the present study demonstrated that UA may be used as a potential therapeutic agent for complications of sepsis, especially for sepsis-induced AKI.

Assessing the effect of Mentha longifolia essential oils on COX-2 expression in animal model of sepsis induced by caecal ligation and puncture

Context: Mentha longifolia L. (Lamiaceae), a traditional Iranian plant, possesses antimicrobial and antioxidant activities.Objective: We investigated the potential protective effects of M. longifolia essential oils (E.Os) on caecal ligation and puncture (CLP) induced liver injury.Materials and methods: Wistar Albino rats (n = 50) were grouped as follows: (1) a laparotomy group (LAP); (2) a CLP group (CLP); (3) the treatment groups received orally the E.Os (50 and 100 mg/kg b.w) and indomethacin (2 mg/kg b.w) for 2 weeks. The oxidative stress parameters, liver enzymes and prostaglandin E2 (PGE2) level were measured in liver and plasma tissues. The liver was also harvested for the real time PCR of cyclooxygenase (COX-2) expression following histopathological examinations.Results: The results indicated that the CLP operation significantly increased lipid peroxidation (LP) [1.79-fold], myeloperoxidase (MPO) [2.76-fold], PGE2 [1.56-fold] besides plasma aspartate aminotransferase (AST) [2.4-fold] and alanine aminotransferase (ALT) activities [2.22-fold], while, markedly reduced glutathione (GSH) [0.63-fold] and ferric reducing ability of plasma (FRAP) levels [0.63-fold]. Even COX2 expression significantly increased in the CLP group as compared to the LAP group. Treatments of rats with the E.Os could return all the hepatic and plasma biomarkers to the normal levels. These results were further confirmed by pathological examination on liver indicating that E.Os could successfully improve the CLP-induced liver injuries.Discussion and conclusions: Our findings suggest that E.Os is able to protect liver injuries against sepsis via modulating the oxidative stress parameters concomitant with the suppression of inflammatory reactions such as PGE2 and COX-2.

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS-induced BBB disruption.

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine-1-phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context-dependent manner. We utilized lipopolysaccharide (LPS)-injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood-brain barrier (BBB) breakdown invivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMVs), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMVs and brain tissue. Primary mouse brain microvascular endothelial cells (MBMECs) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER) as a measure of permeability invitro. We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMVs, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP1) and S1P phosphatase 1 on the other hand, as well as a down-regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMECs did not alter TEER, but serum from LPS-treated mice lead to a breakdown of the barrier compared to serum from vehicle-treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis. Read the Editorial Highlight for this article on page 115. Cover Image for this Issue: doi. 10.1111/jnc.14161.

Impact of unfractionated heparin on serum and liver tissue expression of heparanase in the liver injury of mice with sepsis

To investigate the effect of unfractionated heparin on the expression of serum and liver tissue heparanase (HPA) in mice with liver injury induced by sepsis. Forty-eight healthy male C57BL/6 mice aged 6-8 weeks were divided into groups according to random number table method. Twenty-four septic mice models (CLP group) were established by cecal ligation and puncture (CLP); the other 24 mice underwent sham operation (sham group), only laparotomy and abdominal closure were performed without ligation. Twelve mice in sham group and CLP group received heparin pretreatment (sham+UFH group, CLP+UFH group), and 8 U heparin unfractionated heparin (diluted to 200 μL) was injected into the tail vein of the mice at 30 minutes and 12 hours after operation respectively. The other 12 mice were injected with the same amount of normal saline. The serum and liver tissues of mice were collected at 4 and 24 hours after CLP. The levels of serum HPA, interleukin (IL-6, IL-1β), tumor necrosis factor-α (TNF-α), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by enzyme linked immunosorbent assay (ELISA). The pathological changes of liver tissue were observed with hematoxylin eosin (HE) staining. The expression of HPA in liver tissue was detected by immunohistochemistry. Compared with the sham group, the levels of serum HPA, IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were increased significantly, and increased further over time. The histopathology examination was performed, and abnormal structure, inflammatory cell infiltration, liver cell necrosis could be found in the tissue. The expression level of HPA in liver tissue was detected by immunohistochemistry, which was increased after CLP. This indicated that the animal model of sepsis was successfully prepared. Compared with CLP group, serum HPA, inflammatory factors and transaminase levels were significantly decreased at 4 hours after operation in group CLP+UFH [HPA (ng/L): 76.72±2.75 vs. 101.55±7.54, IL-6 (ng/L): 51.16±5.68 vs. 63.89±3.26, IL-1β (ng/L): 31.53±2.90 vs. 40.87±2.88,TNF-α (ng/L): 171.76±5.60 vs. 194.62±14.13, ALT (μg/L): 0.26±0.09 vs. 0.62±0.17, AST (μg/L): 1.03±0.22 vs. 1.45±0.08, all P < 0.05]. At 24 hours, it was significantly higher than that of 4 hours, but they were significantly lower than those in CLP group [HPA (ng/L): 125.30±7.80 vs. 302.50±17.81, IL-6 (ng/L): 81.16±4.54 vs. 176.56±5.45, IL-1β (ng/L): 61.13±2.80 vs. 113.73±3.96, TNF-α (ng/L): 328.47±10.79 vs. 599.62±10.20, ALT (μg/L): 0.38±0.17 vs. 0.91±0.26, AST (μg/L): 1.16±0.15 vs. 1.88±0.08, all P < 0.05]. It was shown by HE staining that the edema of liver tissue decreased and inflammatory cell infiltration decreased. It was shown by immunohistochemistry that the expression level of HPA in liver tissue was significantly decreased [A value (×10-3): 2.49±0.93 vs. 6.05±1.22 at 4 hours, 1.86±0.77 vs. 7.55±0.35 at 24 hours, both P < 0.05]. There was no significant difference in indexes between the sham+UFH group and the sham group. The expression of HPA was significantly increased during sepsis in mice. Unfractionated heparin may mitigate liver injury by inhibiting HPA.

Glucose homeostasis in two degrees of sepsis lethality induced by caecum ligation and puncture in mice.

Sepsis is associated with high mortality. Both critically ill humans and animal models of sepsis exhibit changes in their glucose homeostasis, that is, hypoglycaemia, with the progression of infection. However, the relationship between basal glycaemia, glucose tolerance and insulin sensitivity is not well understood. Thus, we aimed to evaluate this glucose homeostasis triad at the late stage of sepsis (24h after surgery) in male Swiss mice subjected to lethal and sublethal sepsis by the caecal ligation and puncture (CLP) model. The percentage of survival 24h after CLP procedure in the Lethal and Sublethal groups was around 66% and 100% respectively. Both Lethal and Sublethal groups became hypoglycaemic in fasting and fed states 24h after surgery. The pronounced fed hypoglycaemia in the Lethal group was not due to worsening anorexic behaviour or hepatic inability to deliver glucose in relation to the Sublethal group. Reduction in insulin sensitivity in CLP mice occurred in a lethality-dependent manner and was not associated with glucose intolerance. Analysis of oral and intraperitoneal glucose tolerance tests, as well as the gastrointestinal motility data, indicated that CLP mice had reduced intestinal glucose absorption. Altogether, we suggest cessation of appetite and intestinal glucose malabsorption are key contributors to the hypoglycaemic state observed during experimental severe sepsis.

Lipopolysaccharide Potentiates Insulin-Driven Hypoglycemic Shock.

Critically ill patients typically present with hyperglycemia. Treatment with conventional insulin therapy (targeting 144-180 mg/dl) improves patient survival; however, intensive insulin therapy (IIT) targeting normal blood glucose levels (81-108 mg/dl) increases the incidence of moderate and severe hypoglycemia, and increases mortality. Septic patients are especially prone to IIT-induced hypoglycemia, but the mechanism remains unknown. Here, we show that codelivery of insulin with otherwise sublethal doses of LPS induced hypoglycemic shock in mice within 1-2 h. LPS impaired clearance of insulin, which amplified insulin receptor signaling. These effects were mediated by caspase-11, TLR4, and complement, each of which trigger eicosanoid production that potentiates insulin signaling. Finally, in an animal model of sepsis, we observed that Salmonella typhimurium-infected mice exhibited simultaneous impaired insulin clearance coexisting with insulin resistance. Our results raise the possibility that septic patients have impaired insulin clearance, which could increase their susceptibility to hypoglycemia during IIT, contraindicating its use.

Expression of Toll-like receptors (TLRs) in the lungs of an experimental sepsis mouse model.

BackgroundSepsis is a condition characterized by high mortality rates and often accompanied by multiple-organ dysfunction. During sepsis, respiratory system may be affected and possibly result in acute respiratory distress syndrome (ARDS). Toll-like receptors (TLRs), as a first line defense against invading pathogens, seem to be highly expressed in septic states. Therefore, expression of TLRs in the lungs of a sepsis animal model could indicate the involvement of the respiratory system and appear as a severity index of the clinical course.Materials and methodsA total of 72 C57BL/6J mice, aged 12–14 weeks, were studied. The animals were divided into 3 sepsis (S) groups (24h, 48h and 72h) and 3 control (C) groups (24h, 48h and 72h), each consisting of 12 mice. The S-groups were subjected to cecal ligation and puncture (CLP) while the C-groups had a sham operation performed. Blood samples were drawn from all groups. Total blood count analysis was performed along with the measurement of certain biochemical markers. Additionally, lung tissues were harvested and the expression of TLRs, namely TLR 2, TLR 3, TLR 4 and TLR 7 were evaluated by means of immunofluorescence (IF) and qRT-PCR (quantitative-Polymerase Chain Reaction). Statistical analysis was performed by using one-way ANOVA followed by student t-test. Results were considered statistically significant when p<0.05.ResultsWBCs and lymphocytes were decreased in all S-groups compared to the corresponding C-groups (p<0.05), while RBCs showed a gradual decline in S-groups with the lowest levels appearing in the S72 group. Only, monocytes were higher in S-groups, especially between S48-C48 (p<0.05) and S72-C72 (p<0.05). Creatinine, IL-10 and IL-6 levels were significantly increased in the S-groups compared to the corresponding C-groups (S24 vs C24, S48 vs C48 and S72 vs C72, p<0.05). IF showed that expression of TLRs 2, 3, 4 and 7 was increased in all S-groups compared to the time-adjusted C-groups (p<0.05). Similarly, qRT-PCR revealed that expression of all TLRs was higher in all S-groups compared to their respective C-groups in both lungs and intestine (p<0.05). Comparing lung and intestinal tissues from S-groups, TLRs 2 and 4 were found increased in the lung at 24, 48 and 72 hours (p<0.05), whereas TLR 3 was higher in the intestine at all time points examined (p<0.05). Finally, TLR 7 levels were significantly higher in the intestinal tissues at 24 hours (p<0.0001), while lungs predominated at 48 hours (p<0.0001).ConclusionTLRs seem to be highly expressed in the lungs of septic mice, therefore suggesting a potential role in the pathogenesis of ARDS during sepsis. While more studies need to be conducted in order to completely understand the underlying mechanisms, TLRs may represent a promising target for establishing novel therapeutic strategies in the treatment of sepsis.

Emerging roles of mitochondria and autophagy in liver injury during sepsis.

Recent research indicates crucial roles of autophagy during sepsis. In animal models of sepsis induced by cecal ligation and puncture (CLP) or the systemic administration of lipopolysaccharides (LPS), autophagy is implicated in the activation and/or damage of various cells/organs, such as immune cells, heart, lung, kidney, and liver. Since sepsis is associated with an increased production of pro- as well as anti-inflammatory cytokines, it has long been considered that hypercytokinemia is a fetal immune response leading to multiple organ failure (MOF) and mortality of humans during sepsis. However, a recent paradigm illuminates the crucial roles of mitochondrial dysfunction as well as the perturbation of autophagy in the pathogenesis of sepsis. In the livers of animal models of sepsis, autophagy is involved in the elimination of damaged mitochondria to prevent the generation of mitochondrial ROS and the initiation of the mitochondrial apoptotic pathway. In addition, many reports now indicate that the role of autophagy is not restricted to the elimination of hazardous malfunctioning mitochondria within the cells; autophagy has been shown to be involved in the regulation of inflammasome activation and the release of cytokines as well as other inflammatory substances. In this review, we summarize recent literature describing the versatile role of autophagy and its possible implications in the pathogenesis of sepsis in the liver.

Dimethyl Fumarate Modulates Oxidative Stress and Inflammation in Organs After Sepsis in Rats.

Sepsis is defined as life-threatening organ dysfunction induced by a disrupted host response to infecting pathogens. Evidences suggest that oxidative stress is intrinsically related to sepsis progression. Dimethyl fumarate (DMF) is a novel oral therapeutic agent with anti-oxidant properties which exerts protective effects through activation of nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2). Thus, the aim of this study is to evaluate the effect of DMF in different organs of rats submitted to an animal model of sepsis. Adult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) procedure and sham-operated rats was considered control group. The experimental groups were divided into sham+vehicle, sham+DMF, sham+NAC, CLP+vehicle, CLP+DMF, and CLP+NAC. Rats were treated by oral gavage with DMF immediately after and 12h after surgery, or NAC (s.c.) at 3, 6, and 12h after surgery. Twenty-four hours after sepsis induction, neutrophil infiltration, nitrite/nitrate concentrations, oxidative damage to lipids and proteins, superoxide dismutase (SOD), and catalase (CAT) activities were evaluated in the heart, liver, lung, and kidney. Septic animals presented increased neutrophil infiltration, NO metabolism, oxidative damage to lipids and proteins, and decreases of SOD and CAT activities, mainly in the heart, liver, and lung, while DMF-treated animals showed significant reduction in neutrophil infiltration, NO metabolism, and oxidative damage followed by increased SOD and CAT activities. DMF is effective in preventing oxidative stress and inflammation in rats 24h after sepsis induction.