Cecal Ligation And Puncture Animals Research Articles

EARLY BLOOD LACTATE AS A BIOMARKER FOR CARDIOVASCULAR COLLAPSE IN EXPERIMENTAL SEPSIS.

Cecal ligation and puncture (CLP) is the gold standard model for studying septic shock, which is characterized by hypotension and hyporeactivity to vasoconstrictors. However, approximately 30% of CLP animals do not exhibit cardiovascular changes, requiring more replicates because of the high variability of the model. Therefore, biomarkers enabling the early prediction of cardiovascular collapse in sepsis would greatly benefit sepsis nonclinical studies, refining experimental models and improving clinical translation. Thus, this study aimed to test whether the early increase in lactate levels could predict hypotension and hyporesponsiveness to vasoconstrictors in a rat model of sepsis. Male and female Wistar rats were subjected to CLP or sham procedure. Tail blood lactate was measured 6, 12, and 24 h after surgery. Then, inflammatory, biochemical, and hemodynamic parameters were evaluated. Rats subjected to CLP developed hypotension, hyporesponsiveness to vasoconstrictors, an intense inflammatory process, and increased plasma markers of organ dysfunction. By using receiver operating characteristics curve analysis, we have established that a lactate value of 2.45 mmol/L can accurately discriminate between a rat exhibiting a normal vasoconstrictive response and a vasoplegic rat with 84% accuracy (area under the curve: 0.84; confidence interval [CI]: 0.67-1.00). The sensitivity, which is the ability to identify a diseased rat (true positive), was 75% (CI: 41-95), and the true negative rate was 81% (CI: 57-93). Therefore, early measurement of lactate levels in sepsis could serve as a valuable biomarker for distinguishing vasoplegic rats from those exhibiting normal vasoconstrictive responses.

Moderate exercise-induced dynamics on key sepsis-associated signaling pathways in the liver

BackgroundThere is a clear relationship between quantitative measures of fitness (e.g., VO2 max) and outcomes after surgical procedures. Whether or not fitness is a modifiable risk factor and what underlying biological processes drive these changes are not known. The purpose of this study was to evaluate the moderate exercise training effect on sepsis outcomes (survival) as well as the hepatic biological response. We chose to study the liver because it plays a central role in the regulation of immune defense during systemic infection and receives blood flow directly from the origin of infection (gut) in the cecal ligation and puncture (CLP) model.MethodsWe randomized 50 male (♂) and female (♀) Sprague–Dawley rats (10 weeks, 340 g) to 3 weeks of treadmill exercise training, performed CLP to induce polymicrobial “sepsis,” and monitored survival for five days (Part I). In parallel (Part II), we randomized 60 rats to control/sedentary (G1), exercise (G2), exercise + sham surgery (G3), CLP/sepsis (G4), exercise + CLP [12 h (G5) and 24 h (G6)], euthanized at 12 or 24 h, and explored molecular pathways related to exercise and sepsis survival in hepatic tissue and serum.ResultsThree weeks of exercise training significantly increased rat survival following CLP (polymicrobial sepsis). CLP increased inflammatory markers (e.g., TNF-a, IL-6), which were attenuated by exercise. Sepsis suppressed the SOD and Nrf2 expression, and exercise before sepsis restored SOD and Nrf2 levels near the baseline. CLP led to increased HIF1a expression and oxidative and nitrosative stress, the latter of which were attenuated by exercise. Haptoglobin expression levels were increased in CLP animals, which was significantly amplified in exercise + CLP (24 h) rats.ConclusionsModerate exercise training (3 weeks) increased the survival in rats exposed to CLP, which was associated with less inflammation, less oxidative and nitrosative stress, and activation of antioxidant defense pathways.Graphical abstract

Early effects of bone marrow-derived mononuclear cells on lung and kidney in experimental sepsis

BackgroundIn experimental sepsis, functional and morphological effects of bone marrow-derived mononuclear cell (BMDMC) administration in lung tissue have been evaluated 1 and 7 days after therapy. However, to date no study has evaluated the early effects of BMDMCs in both lung and kidney in experimental polymicrobial sepsis. Material and methodsTwenty-five female C57BL/6 mice were randomly divided into the following groups: 1) cecal ligation and puncture (CLP)-induced sepsis; and 2) Sham (surgical procedure without CLP). After 1 h, CLP animals received saline (NaCl 0.9%) (CLP-Saline) or 106 BMDMCs (CLP-Cell) via the jugular vein. At 6, 12, and 24 h after saline or BMDMC administration, lungs and kidneys were removed for histology and molecular biology analysis. ResultsIn lungs, CLP-Saline, compared to Sham, was associated with increased lung injury score (LIS) and keratinocyte chemoattractant (KC) mRNA expression at 6, 12, and 24 h. BMDMCs were associated with reduced LIS and KC mRNA expression regardless of the time point of analysis. Interleukin (IL)− 10 mRNA content was higher in CLP-Cell than CLP-Saline at 6 and 24 h. In kidney tissue, CLP-Saline, compared to Sham, was associated with tubular cell injury and increased neutrophil gelatinase-associated lipocalin (NGAL) levels, which were reduced after BMDMC therapy at all time points. Surface high-mobility-group-box (HMGB)− 1 levels were higher in CLP-Saline than Sham at 6, 12, and 24 h, whereas nuclear HMGB-1 levels were increased only at 24 h. BMDMCs were associated with decreased surface HMGB-1 and increased nuclear HMGB-1 levels. Kidney injury molecule (KIM)− 1 and IL-18 gene expressions were reduced in CLP-Cell compared to CLP-Saline at 12 and 24 h. ConclusionIn the present experimental polymicrobial sepsis, early intravenous therapy with BMDMCs was able to reduce lung and kidney damage in a time-dependent manner. BMDMCs thus represent a potential therapy in well-known scenarios of sepsis induction. PurposeTo evaluate early bone marrow-derived mononuclear cell (BMDMC) therapy on lung and kidney in experimental polymicrobial sepsis. MethodsTwenty-five female C57BL/6 mice were randomly divided into the following groups: cecal ligation and puncture (CLP)-induced sepsis; and sham (surgical procedure without CLP). After 1 h, CLP animals received saline (CLP-saline) or 106 BMDMCs (CLP-cell) via the jugular vein. Lungs and kidneys were evaluated for histology and molecular biology after 6, 12, and 24 h. ResultsIn lungs, BMDMCs reduced the lung injury score and keratinocyte chemoattractant mRNA expression regardless of the time point of analysis; interleukin-10 mRNA content was higher in CLP-cell than CLP-saline at 6 and 24 h. In kidneys, BMDMCs reduced neutrophil gelatinase-associated lipocalin levels at all time points. BMDMCs decreased surface high mobility group box (HMGB)− 1 but increased nuclear HMGB-1 levels. ConclusionEarly BMDMC therapy reduced lung and kidney damage in a time-dependent manner.

Comparison of Single and Repeated Dosing of Anti-Inflammatory Human Umbilical Cord Mesenchymal Stromal Cells in a Mouse Model of Polymicrobial Sepsis

SummaryMesenchymal stromal cells (MSCs) ameliorate pre-clinical sepsis and sepsis-associated acute kidney injury (SA-AKI) but clinical trials of single-dose MSCs have not indicated robust efficacy. This study investigated immunomodulatory effects of a novel MSC product (CD362-selected human umbilical cord-derived MSCs [hUC-MSCs]) in mouse endotoxemia and polymicrobial sepsis models. Initially, mice received intra-peritoneal (i.p.) lipopolysaccharide (LPS) followed by single i.p. doses of hUC-MSCs or vehicle. Next, mice underwent cecal ligation and puncture (CLP) followed by intravenous (i.v.) doses of hUC-MSCs at 4 h or 4 and 28 h. Analyses included serum/plasma assays of biochemical indices, inflammatory mediators and the AKI biomarker NGAL; multi-color flow cytometry of peritoneal macrophages (LPS) and intra-renal immune cell subpopulations (CLP) and histology/immunohistochemistry of kidney (CLP). At 72 h post-LPS injections, hUC-MSCs reduced serum inflammatory mediators and peritoneal macrophage M1/M2 ratio. Repeated, but not single, hUC-MSC doses administered at 48 h post-CLP resulted in lower serum concentrations of inflammatory mediators, lower plasma NGAL and reversal of sepsis-associated depletion of intra-renal T cell and myeloid cell subpopulations. Hierarchical clustering analysis of all 48-h serum/plasma analytes demonstrated partial co-clustering of repeated-dose hUC-MSC CLP animals with a Sham group but did not reveal a distinct signature of response to therapy. It was concluded that repeated doses of CD362-selected hUC-MSCs are required to modulate systemic and local immune/inflammatory events in polymicrobial sepsis and SA-AKI. Inter-individual variability and lack of effect of single dose MSC administration in the CLP model are consistent with observations to date from early-phase clinical trials.Graphical

Pharmacological Effects of Marine-Derived Enterococcus faecium EA9 against Acute Lung Injury and Inflammation in Cecal Ligated and Punctured Septic Rats.

Microorganisms obtained from the marine environment may represent a potential therapeutic value for multiple diseases. This study explored the possible protective role of marine-derived potential probiotic Enterococcus faecium EA9 (E. faecium) against pulmonary inflammation and oxidative stress using the cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Animals were pretreated with E. faecium for 10 days before either sham or CLP surgeries. Animals were sacrificed 72 hours following the surgical intervention. The histological architecture of lung tissues was evaluated as indicated by the lung injury score. In addition, the extend of pulmonary edema was determined as wet/dry weight ratio. The inflammatory cytokines were estimated in lung tissues, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) using the enzyme-linked-immunosorbent-assay (ELISA) technique. Moreover, markers for lipid peroxidation such as thiobarbituric acid reaction substances (TBARs), and endogenous antioxidants, including reduced glutathione (GSH) were determined in lung tissues. Finally, the enzymatic activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were assayed in the lungs. Pretreatment with E. faecium markedly attenuated CLP-induced lung injury and pulmonary edema. Markers for inflammation, including TNF-α, IL-6, and IL-1β were augmented in the lung tissues of CLP animals, while E. faecium ameliorated their augmented levels. E. faecium pretreatment also restored the elevated TBARS levels and the prohibited CAT, SOD, and GPx enzymatic activities in CLP animals. GSH levels were corrected by E. faecium in CLP animals. The inflammatory and lipid peroxidation mediators were positively correlated, while antioxidant enzymatic activities were negatively correlated with CLP-induced lung injury and pulmonary edema. Collectively, marine-derived Enterococcus faecium EA9 might be considered as a prospective therapeutic tool for the management of pulmonary dysfunction associated with sepsis.

MITOCHONDRIA ISOLATED FROM MESENCHYMAL STROMAL CELLS REDUCE LUNGAND DISTAL ORGAN INJURY IN EXPERIMENTAL SEPSIS

<h3>Background</h3> Although mesenchymal stromal cells (MSC) administration has demonstrated beneficial effects in experimental sepsis, cell-free therapeutics based on biologically active products from MSCs have been proposed to circumvent inherent risks related to whole-cell transplantation. As mitochondria transfer has emerged as one of the key mechanisms by which MSCs induce therapeutic actions, we asked whether MSC-isolated mitochondria could reduce lung, kidney, and liver injury, thus improving survival in experimental sepsis. <h3>Methods</h3> Experimental sepsis was induced by cecal ligation and puncture (CLP), while sham-operated animals were used as control. Twenty-four hours after surgery, CLP and Sham animals were further randomized to receive saline or MSC-isolated mitochondria (MT, from 3 × 106 MSCs) intravenously. After additional 24 h, survival rate, peritoneal bacterial load, lung mechanics and histology, kidney and liver injury, kidney function, as well as expression levels of selected mediators in tissue homogenates were analyzed. Moreover, the uptake and effects mitochondria on oxygen consumption rate (OCR) and reactive oxygen species (ROS) production of lung epithelial and endothelial cells were evaluated in vitro. <h3>Results</h3> In vitro exposure of lung epithelial and endothelial cells from CLP animals to MSC-isolated mitochondria resulted in mitochondrial uptake by endothelial cells and restored OCR and reduced total ROS production. Systemic administration of MSC-isolated mitochondria led to reduction of bacterial load, static lung elastance, alveolar collapse, neutrophil count, collagen and elastic fiber content as well as expression of interleukin (IL)-1β, keratinocyte chemoattractant, indoleamine 2,3-dioxygenase (IDO)-2 and programmed cell death protein 1 (PD-1) in lung tissue, while increasing KGF levels and survival rate in CLP-induced sepsis. Mitochondria therapy also reduced kidney and liver injury, decreasing plasma creatinine levels, mRNA expression of IL-18 in kidney, IL-6, IDO-2 and PD-1 in liver and increasing mRNA levels of nuclear factor erythroid 2-related factor-2 and superoxide dismutase-2 in kidney and IL-10 in liver. <h3>Conclusions</h3> Mitochondria therapy enabled the recovery of host mitochondrial function and reduced inflammation and oxidative stress, thus decreasing lung, liver, and kidney injury and increasing survival rate in CLP-induced sepsis.

Systemic Effects Induced by Hyperoxia in a Preclinical Model of Intra-abdominal Sepsis.

Supplemental oxygen is a supportive treatment in patients with sepsis to balance tissue oxygen delivery and demand in the tissues. However, hyperoxia may induce some pathological effects. We sought to assess organ damage associated with hyperoxia and its correlation with the production of reactive oxygen species (ROS) in a preclinical model of intra-abdominal sepsis. For this purpose, sepsis was induced in male, Sprague-Dawley rats by cecal ligation and puncture (CLP). We randomly assigned experimental animals to three groups: control (healthy animals), septic (CLP), and sham-septic (surgical intervention without CLP). At 18 h after CLP, septic (n = 39), sham-septic (n = 16), and healthy (n = 24) animals were placed within a sealed Plexiglas cage and randomly distributed into four groups for continuous treatment with 21%, 40%, 60%, or 100% oxygen for 24 h. At the end of the experimental period, we evaluated serum levels of cytokines, organ damage biomarkers, histological examination of brain and lung tissue, and ROS production in each surviving animal. We found that high oxygen concentrations increased IL-6 and biomarkers of organ damage levels in septic animals, although no relevant histopathological lung or brain damage was observed. Healthy rats had an increase in IL-6 and aspartate aminotransferase at high oxygen concentration. IL-6 levels, but not ROS levels, are correlated with markers of organ damage. In our study, the use of high oxygen concentrations in a clinically relevant model of intra-abdominal sepsis was associated with enhanced inflammation and organ damage. These findings were unrelated to ROS release into circulation. Hyperoxia could exacerbate sepsis-induced inflammation, and it could be by itself detrimental. Our study highlights the need of developing safer thresholds for oxygen therapy.

Prolonged Soluble Epoxide Hydrolase Reactivity in Brain Endothelial Cells Is Associated with Long Cognitive Deficits in Sepsis.

Sepsis-associated encephalopathy (SAE) is known to cause long-term cognitive deficits which are related to sustained microglial activation, but the mechanisms are unclear. Recently, studies have shown soluble epoxide hydrolase (sEH) affects the chronic cognitive function or participates in long-term neuropsychiatric illness. We hypothesized that sEH may be involved in the long-term cognitive deficits of SAE. Male C57BL/6 mice were subjected to cecal ligation and puncture (CLP) and were administered vehicle or sEH inhibitor TPPU. CLP induced prolonged endothelial sEH reactivity and sustained activation of microglia in close vicinity to blood vessels at 14days. We also observed that persistent loss of endothelial BBB function at 14days following CLP. However, TPPU-treated septic mice exhibited improved BBB function and declined neuro-inflammation. We confirmed these beneficial effects in vitro, which indicated TPPU resulted in a significant improvement in IL-1β-induced loss of BBB integrity on hCMEC/D3 cell monolayers. Animals were also given a behavior test at 14days after CLP. Mice showed normal basal locomotor activity in the open field compared with sham-operated animals, but performed fewer entries to the center zone, indicating increased anxiety-like behavior as avoidance of the center. TPPU-treated CLP mice showed normal crossing into the center zone during an open-field test and improved recovery of the ability to learn the novel object recognition (NOR) task compared with saline-treated CLP animals. Our data indicated that prolonged sEH reactivity in brain endothelial cells is associated with long cognitive deficits in sepsis. sEHIs such as TPPU can improve the endothelial barrier function and decrease CLP-induced long-term encephalopathy, at least in part, through anti-inflammatory effects.

P717Glucagon-like peptide 1 (GLP-1) improves endothelial dysfunction and vascular inflammation in polymicrobial sepsis induced by cecal ligation and puncture (CLP)

Abstract Objective Sepsis causes severe hypotension, accompanied by high mortality in the setting of septic shock. LEADER, SUSTAIN-6 and other clinical trials revealed cardioprotective and anti-inflammatory properties of GLP-1 analogs like Liraglutide (Lira). We already demonstrated improved survival by amelioration of disseminated intravasal coagulation (DIC) in lipopolysaccharide (LPS)-induced endotoxemia by inhibition of the GLP-1 degrading enzyme dipeptidylpeptidase-4 (DPP-4). With the present study we aim to investigate the mechanism of protective effects of the GLP-1 analog Lira and the DPP4 inhibitor Linagliptin (Lina) in the clinically relevant sepsis model cecal ligation and puncture (CLP). Methods C57/BL6j and endothelial cell-specific GLP-1 receptor knockout mice (Cdh5crexGLP-1rfl/flmice) were used and sepsis was induced by cecal ligation and puncture (CLP). DPP4 inhibitor (Lina, 5mg/kg/d; 3 days) and GLP-1 analog (Lira, 200μg/kg/d; 3 days) were applied subcutaneously. Aortic vascular function was tested by isometric tension recording. Aorta and heart tissue was used for Western blotting, dot blot and qRT-PCR. Endogenous GLP-1 (7–36 and 9–36) and insulin was determined by ELISA. Blood samples were collected for examination of cell count, oxidative stress and glucose levels. Results Body temperature was increased by CLP and normalized by Lina and Lira. Sham- and Lira- but not Lina-treated septic mice showed low blood glucose levels compared to healthy controls. Acetylcholine-induced (endothelium-dependent) vascular relaxation in aorta was impaired by CLP. This was accompanied by vascular inflammation and elevation of IL-6, iNOS, ICAM-1, and TNF-alpha mRNA levels in aortic tissue. Vascular, cardiac and whole blood oxidative stress were increased by CLP. Furthermore, we detected higher levels of IL-6, 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-NHE) in plasma of CLP animals. Lina and Lira reduced oxidative stress and vascular inflammation, which was accompanied by improved endothelial function. In addition, CLP treatment in endothelial specific knockout mice of the GLP-1r strongly induced mortality compared to WT mice, with the effect being strongest in the Lira-treated group. Conclusion The present study demonstrates that Lina (DPP4 inhibitor) and the GLP-1 analog Lira ameliorate sepsis-induced endothelial dysfunction by reduction of vascular inflammation and oxidative stress. Clinical trials like LEADER and SUSTAIN-6 proved that GLP-1 analogs like Lira have cardioprotective effects in T2DM patients. The present study, performed in a clinically relevant model of polymicrobial sepsis, reveals that the known cardioprotective effects of GLP-1 might be translated to other diseases which affect the cardiovascular system like sepsis, underlining the potent anti-inflammatory effects of GLP-1 analogs.

Influence of autophagy on acute kidney injury in a murine cecal ligation and puncture sepsis model

The role of autophagy in the maintenance of renal homeostasis during sepsis is not well understood. We therefore aimed to determine the influence of autophagy on kidney during sepsis using a murine sepsis model, i.e. cecal ligation and puncture (CLP). In CLP treated animals, the number of autolysosomes observed by electron microscopy increased over time. The number of autophagosomes in CLP animals decreased relative sham operated controls at 24 hrs after CLP, indicating that autophagy flux is already diminishing by that time. Moreover, CLP induced an increase in LC3-II/LC3-I ratio at 6–8 hrs, demonstrated in western blots, as well as an increase in GFP-LC3 dots at 6–8 hrs and 24 hrs, using immunofluorescence and anti-LC3 and LAMP1 antibodies on tissue sections from GFP-LC3 transgenic mice. LC3-II/LC3-I ratio and the number of co-localized GFP-LC3 dots and LAMP1 signals (GFP LC3 + LAMP1 dots) in CLP mice at 24 hrs were significantly reduced compared with data obtained at 6–8 hrs. Notably, acceleration of autophagy by rapamycin resulted in improvement of renal function that was associated with improvement in the histologic severity of tubular epithelial injury in CLP treated animals. Autophagy in the kidney was significantly slowed in the kidney during the acute phase of sepsis; nonetheless, autophagy in kidney appears to play a protective role against sepsis.

Abstract P551: Platelet Activation is Associated with Pulmonary Edema and Renal Injury in a Rat Model of Polymicrobial Sepsis

Sepsis, life-threatening organ dysfunction due to a dysregulated host response to infection, is positively correlated with platelet activation. Furthermore, clinical studies have also shown that platelet activation is associated with sepsis severity, suggesting a role for platelets in sepsis pathophysiology. Despite this correlation, the underlying mechanisms by which activated platelets contribute to sepsis are under investigated. In preliminary studies, we set out to determine if platelet activation is associated with multi-organ dysfunction and injury in a rat model of chronic polymicrobial abdominal sepsis. Sepsis was induced via cecal ligation and puncture (CLP) followed by cecum removal 24 hours post-CLP. At 72 hours post-CLP, blood, urine, and tissues were collected for analysis. Platelet activation was measured via flow cytometry. Lung wet/dry ratio and plasma creatinine were measured to assess lung edema and renal injury, respectively. Platelet activation doubled in CLP rats versus Sham rats. Activated platelets increased from 3.8±1.7% of the gated population in Sham animals (n=5) to 9.2±1.9% of the gated population in CLP animals (n=5; p=0.07). Lung wet/dry ratio significantly increased from 3.9±0.2 in Sham (n=8) to 6.7±1 in CLP rats (n=8; p&lt;0.05). Furthermore, plasma creatinine increased by 33% from 0.55±0.3 mg/dL in Sham animals (n=6) to 0.73±0.06 mg/dL in CLP rats (n=8; p&lt;0.05), indicating a decrease in renal function. These data demonstrate, for the first time, an increase in platelet activation in response to CLP, and identifies an association of activated platelets with pulmonary edema and reduced renal function in the cecal ligation and puncture rat model of abdominal polymicrobial sepsis. Future studies will investigate the underlying mechanisms by which activated platelets contribute to multi-organ dysfunction and injury in sepsis.

494 - Effects of low doses of esmolol on cardiac and vascular function in experimental septic shock

Administration of a selective β1-blocker, such as esmolol, in human septic shock has demonstrated cardiovascular protective effects related to heart rate reduction. Certain experimental data also indicate that esmolol exerts systemic anti-inflammatory and beneficial effects on vascular tone. Thus, the present study aimed to determine whether a non-chronotropic dose of esmolol maintains its protective cardiovascular and anti-inflammatory effects in experimental septic shock. Four hours after cecal ligation and puncture (CLP), Wistar male rats were randomly allocated to the following groups (n = 8): CLP, CLP + E-1 (esmolol: 1 mg.kg−1.h−1), CLP + E-5 (esmolol: 5 mg.kg−1.h−1), CLP + E-18 (esmolol: 18 mg.kg−1.h−1). An additional eight rats underwent sham operation. All rats received a continuous infusion of saline, analgesic and antibiotics 4 hours after the surgery. Assessment at 18 hours included in vivo cardiac function assessed by echocardiography and ex vivo vasoreactivity assessed by myography. Circulating cytokine levels (IL-6 and IL-10) were measured by ELISA. Cardiac and vascular protein expressions of p-NF-κB, IκBα, iNOS, p-AKT/AKT and p-eNOS/eNOS were assessed by western blotting. CLP induced tachycardia, hypotension, cardiac output reduction, hyperlactatemia and vascular hypo-responsiveness to vasopressors. Compared to CLP animals, heart rate was unchanged in CLP + E-1 and CLP + E-5 but was reduced in CLP + E-18. Stroke volume, cardiac output, mean arterial pressure and lactatemia were improved in CLP + E-1 and CLP + E-5, while vascular responsiveness to phenylephrine was only improved in CLP + E-5 and CLP + E-18. Plasma IL-6 levels were decreased in all esmolol groups. p-NF-κB was decreased in both cardiac and vascular tissues in CLP + E-5 and CLP + E-18. In experimental septic shock, low doses of esmolol still improved cardiac function and vasoreactivity. These benefits appear to be associated with a modulation of inflammatory pathways.

Effects of low doses of esmolol on cardiac and vascular function in experimental septic shock.

BackgroundAdministration of a selective β1-blocker, such as esmolol, in human septic shock has demonstrated cardiovascular protective effects related to heart rate reduction. Certain experimental data also indicate that esmolol exerts systemic anti-inflammatory and beneficial effects on vascular tone. Thus, the present study aimed to determine whether a non-chronotropic dose of esmolol maintains its protective cardiovascular and anti-inflammatory effects in experimental septic shock.MethodsFour hours after cecal ligation and puncture (CLP), Wistar male rats were randomly allocated to the following groups (n = 8): CLP, CLP + E-1 (esmolol: 1 mg.kg−1.h−1), CLP + E-5 (esmolol: 5 mg.kg−1.h−1), CLP + E-18 (esmolol: 18 mg.kg−1.h−1). An additional eight rats underwent sham operation. All rats received a continuous infusion of saline, analgesic and antibiotics 4 hours after the surgery. Assessment at 18 hours included in vivo cardiac function assessed by echocardiography and ex vivo vasoreactivity assessed by myography. Circulating cytokine levels (IL-6 and IL-10) were measured by ELISA. Cardiac and vascular protein expressions of p-NF-κB, IκBα, iNOS, p-AKT/AKT and p-eNOS/eNOS were assessed by western blotting.ResultsCLP induced tachycardia, hypotension, cardiac output reduction, hyperlactatemia and vascular hypo-responsiveness to vasopressors. Compared to CLP animals, heart rate was unchanged in CLP + E-1 and CLP + E-5 but was reduced in CLP + E-18. Stroke volume, cardiac output, mean arterial pressure and lactatemia were improved in CLP + E-1 and CLP + E-5, while vascular responsiveness to phenylephrine was only improved in CLP + E-5 and CLP + E-18. Plasma IL-6 levels were decreased in all esmolol groups. p-NF-κB was decreased in both cardiac and vascular tissues in CLP + E-5 and CLP + E-18.ConclusionIn experimental septic shock, low doses of esmolol still improved cardiac function and vasoreactivity. These benefits appear to be associated with a modulation of inflammatory pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-016-1580-2) contains supplementary material, which is available to authorized users.

Involvement of Central Endothelin ETA and Cannabinoid CB1 Receptors and Arginine Vasopressin Release in Sepsis Induced by Cecal Ligation and Puncture in Rats.

We previously reported that endothelin-1 (ET-1) reduced the frequency of spontaneous excitatory currents in vasopressinergic magnocellular cells through the activation of endothelin ETA receptors in rat brain slices. This effect was abolished by a cannabinoid CB1 receptor antagonist, suggesting the involvement of endocannabinoids. The present study investigated whether the blockade of ETA or CB1 receptors during the phase of increased levels of ET-1 after severe sepsis increases the survival rate of animals concomitantly with an increase in plasma arginine vasopressin (AVP) levels. Sepsis was induced in male Wistar rats by cecal ligation and puncture (CLP). Treatment with the CB1 receptor antagonist rimonabant (Rim; 10 and 20 mg/kg, orally) 4 h after CLP (three punctures) significantly increased the survival rate compared with the CLP per vehicle group. Intracerebroventricular treatment with the ETA receptor antagonist BQ123 (100 pmol) or with Rim (2 μg) 4 and 8 h after CLP but not the ETB receptor antagonist BQ788 (100 pmol), also significantly improved the survival rate. Sham-operated and CLP animals that were treated with Rim had significantly lower core temperature than CLP animals. However, oral treatment with Rim did not change bacterial count in the peritoneal exudate, neutrophil migration to the peritoneal cavity, leucopenia or increased plasma interleukin-6 levels induced by CLP. Both Rim and BQ123 also increased AVP levels 12 h after CLP. The blockade of central CB1 and ETA receptors in the late phase of sepsis increased the survival rate, reduced body temperature and increased the circulating AVP levels.

Delayed Treatment with Sodium Hydrosulfide Improves Regional Blood Flow and Alleviates Cecal Ligation and Puncture (CLP)-Induced Septic Shock.

Cecal ligation and puncture (CLP)-induced sepsis is a serious medical condition, caused by a severe systemic infection resulting in a systemic inflammatory response. Recent studies have suggested the therapeutic potential of donors of hydrogen sulfide (H2S), a novel endogenous gasotransmitter and biological mediator in various diseases. The aim of the present study was to assess the effect of H2S supplementation in sepsis, with special reference to its effect on the modulation of regional blood flow. We infused sodium hydrosulfide (NaHS), a compound that produces H2S in aqueous solution (1, 3, or 10 mg/kg/h, for 1 h at each dose level) in control rats or rats 24 h after CLP, and measured blood flow using fluorescent microspheres. In normal control animals, NaHS induced a characteristic redistribution of blood flow, and reduced cardiac, hepatic, and renal blood flow in a dose-dependent fashion. In contrast, in rats subjected to CLP, cardiac, hepatic, and renal blood flow was significantly reduced; infusion of NaHS (1 mg/kg/h and 3 mg/kg/h) significantly increased organ blood flow. In other words, the effect of H2S on regional blood flow is dependent on the status of the animals (i.e., a decrease in blood flow in normal controls, but an increase in blood flow in CLP). We have also evaluated the effect of delayed treatment with NaHS on organ dysfunction and the inflammatory response by treating the animals with NaHS (3 mg/kg) intraperitoneally (i.p.) at 24 h after the start of the CLP procedure; plasma levels of various cytokines and tissue indicators of inflammatory cell infiltration and oxidative stress were measured 6 h later. After 24 h of CLP, glomerular function was significantly impaired, as evidenced by markedly increased (over 4-fold over baseline) blood urea nitrogen and creatinine levels; this increase was also significantly reduced by treatment with NaHS. NaHS also attenuated the CLP-induced increases in malondialdehyde levels (an index of oxidative stress) in heart as well as in liver and myeloperoxidase levels (an index of neutrophil infiltration) in heart and lung. Plasma levels of IL-1β, IL-5, IL-6, TNF-α, and HMGB1 were attenuated by NaHS. Treatment of NaHS at 3 mg/kg i.p. (but not 1 mg/kg or 6 mg/kg), starting 24 h post-CLP, with dosing repeated every 6 h, improved the survival rate in CLP animals. In summary, treatment with 3 mg/kg H2S-when started in a delayed manner, when CLP-induced organ injury, inflammation and blood flow redistribution have already ensued-improves blood flow to several organs, protects against multiple organ failure, and reduces the plasma levels of multiple pro-inflammatory mediators. These findings support the view that H2S donation may have therapeutic potential in sepsis.

Inflammatory Kidney and Liver Tissue Response to Different Hydroxyethylstarch (HES) Preparations in a Rat Model of Early Sepsis.

BackgroundTissue hypoperfusion and inflammation in sepsis can lead to organ failure including kidney and liver. In sepsis, mortality of acute kidney injury increases by more than 50%. Which type of volume replacement should be used is still an ongoing debate. We investigated the effect of different volume strategies on inflammatory mediators in kidney and liver in an early sepsis model.Material and MethodsAdult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) and assigned to three fluid replenishment groups. Animals received 30mL/kg of Ringer’s lactate (RL) for 2h, thereafter RL (75mL/kg), hydroxyethyl starch (HES) balanced (25mL/kg), containing malate and acetate, or HES saline (25mL/kg) for another 2h. Kidney and liver tissue was assessed for inflammation. In vitro rat endothelial cells were exposed to RL, HES balanced or HES saline for 2h, followed by stimulation with tumor necrosis factor-α (TNF-α) for another 4h. Alternatively, cells were exposed to malate, acetate or a mixture of malate and acetate, reflecting the according concentration of these substances in HES balanced. Pro-inflammatory cytokines were determined in cell supernatants.ResultsCytokine mRNA in kidney and liver was increased in CLP animals treated with HES balanced compared to RL, but not after application of HES saline. MCP-1 was 3.5fold (95% CI: 1.3, 5.6) (p<0.01) and TNF-α 2.3fold (95% CI: 1.2, 3.3) (p<0.001) upregulated in the kidney. Corresponding results were seen in liver tissue. TNF-α-stimulated endothelial cells co-exposed to RL expressed 3529±1040pg/mL MCP-1 and 59±23pg/mL CINC-1 protein. These cytokines increased by 2358pg/mL (95% CI: 1511, 3204) (p<0.001) and 29pg/ml (95% CI: 14, 45) (p<0.01) respectively when exposed to HES balanced instead. However, no further upregulation was observed with HES saline. PBS supplemented with acetate increased MCP-1 by 1325pg/mL (95% CI: 741, 1909) (p<0.001) and CINC-1 by 24pg/mL (95% CI: 9, 38) (p<0.01) compared to RL. Malate as well as HES saline did not affect cytokine expression.ConclusionWe identified HES balanced and specifically its component acetate as pro-inflammatory factor. How important this additional inflammatory burden on kidney and liver function is contributing to the sepsis-associated inflammatory burden in early sepsis needs further evaluation.

Inhibition of histone deacetylase 6 restores innate immune cells in the bone marrow in a lethal septic model.

We have previously demonstrated that Tubastatin A, a selective inhibitor of histone deacetylase 6 (HDAC6), improves survival and increases circulating monocyte count and bacterial clearance in a lethal model of cecal ligation and puncture (CLP) in mice. The aim of the present study was to characterize the effects of inhibition of HDAC6 on the bone marrow cell population. C57BL/6J mice were subjected to CLP and, 1 hour later, given an intraperitoneal injection of either Tubastatin A (70 mg/kg) dissolved in DMSO or DMSO alone (n = 9 per group). Sham-operated animals were treated in an identical fashion, without CLP. Forty-eight hours later, bone marrow cells were flushed out from the femurs and tibias. Erythrocytes were lysed, and a single-cell suspension was made for analysis. Cells were washed; blocked with antimouse CD16/32; stained with antimouse B220 PE-Cy7, CD3 APC-eFluor 780, CD11b FITC, Gr-1 PerCP-Cy5.5, and F4/80 Antigen APC; and subjected to flow cytometry. Data were acquired on an LSRII Flow Cytometer (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Flowjo, LLC, Ashland, OR). In comparison with the sham group, CLP animals showed decreased percentage of innate immune cells (CD11b, 62.1% ± 3.1% vs. 32.9% ± 4.9%, p = 0.0025) and macrophages (CD11bF4/80, 44.6% ± 3.4% vs. 19.8% ± 2.6%, p = 0.0002) as well as increased percentage of T lymphocytes (CD3, 1.1% ± 0.2% vs. 3.3% ± 0.4%, p = 0.0082) in the bone marrow 48 hours after CLP. Treatment with Tubastatin A restored the innate immune cells (32.9% ± 4.9% vs. 54.0% ± 4.1%, p = 0.0112) and macrophages (19.8% ± 2.6% vs. 47.1% ± 4.6%, p = 0.0001) and increased the percentage of neutrophils (CD11bGr-1, 28.4% ± 3.9% vs. 48.0% ± 4.0%, p = 0.0075). The percentages of B (B220) and T lymphocytes were not significantly altered by Tubastatin A, compared with the vehicle-treated CLP animals. Selective inhibition of HDAC6 in this lethal septic model restored the innate immune cell and macrophage populations and increased the neutrophil composition in the bone marrow. These results may explain the previously reported beneficial effects of Tubastatin A treatment in a septic model.

Propofol increases morbidity and mortality in a rat model of sepsis.

IntroductionSevere sepsis is associated with approximately 50% mortality and accounts for tremendous healthcare costs. Most patients require ventilatory support and propofol is commonly used to sedate mechanically ventilated patients. Volatile anesthetics have been shown to attenuate inflammation in a variety of different settings. We therefore hypothesized that volatile anesthetic agents may offer beneficial immunomodulatory effects during the course of long-term intra-abdominal sepsis in rats under continuous sedation and ventilation for up to 24 hours.MethodsSham operation or cecal ligation and puncture (CLP) was performed in adult male Wistar rats followed by mechanical ventilation. Animals were sedated for 24 hours with propofol (7 to 20 mg/kg/h), sevoflurane, desflurane or isoflurane (0.7 minimal alveolar concentration each).ResultsSeptic animals sedated with propofol showed a mean survival time of 12 hours, whereas >56% of all animals in the volatile groups survived 24 hours (P <0.001). After 18 hours, base excess in propofol + CLP animals (−20.6 ± 2.0) was lower than in the volatile groups (isoflurane + CLP: -11.7 ± 4.2, sevoflurane + CLP: -11.8 ± 3.5, desflurane + CLP -14.2 ± 3.7; all P <0.03). Plasma endotoxin levels reached 2-fold higher levels in propofol + CLP compared to isoflurane + CLP animals at 12 hours (P <0.001). Also blood levels of inflammatory mediators (tumor necrosis factor-α, interleukin-1β, interleukin-10, CXCL-2, interferon-γ and high mobility group protein-1) were accentuated in propofol + CLP rats compared to the isoflurane + CLP group at the same time point (P <0.04).ConclusionsThis is the first study to assess prolonged effects of sepsis and long-term application of volatile sedatives compared to propofol on survival, cardiovascular, inflammatory and end organ parameters. Results indicate that volatile anesthetics dramatically improved survival and attenuate systemic inflammation as compared to propofol. The main mechanism responsible for adverse propofol effects could be an enhanced plasma endotoxin concentration, leading to profound hypotension, which was unresponsive to fluid resuscitation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0751-x) contains supplementary material, which is available to authorized users.

Interleukin-1 Receptor Antagonist Decreases Hypothalamic Oxidative Stress During Experimental Sepsis.

In our previous work, we demonstrated that the intracerebroventricular (i.c.v.) injection of an interleukin-1 receptor antagonist (IL-1ra) prevented the impairment in vasopressin secretion and increased survival rate in septic rats. Additionally, we saw a reduction in nitric oxide (NO) levels in cerebroventricular spinal fluid (CSF), suggesting that the IL-1ra prevents apoptosis that seems to occur in vasopressinergic neurons. Here, we investigated the effect of IL-1ra pre-treatment on the sepsis-induced increase in oxidative stress markers in the hypothalamus of rats. The animals were pre-treated by an i.c.v. injection of IL-1ra (9nmol) or vehicle (0.01M PBS) before being subjected to cecal ligation and puncture (CLP) or left as control (sham-operation or naive). After 4, 6, and 24h, the animals were decapitated (n = 9/group) and the brain removed for hypothalamic tissue collection. Transcript and protein levels of IL-1, inducible nitric oxide synthase (iNOS), caspase-3, and hypoxia-inducible factor 1-alpha (HIF-1α) were measured by quantitative polymerase chain reaction (qPCR) and western blot, respectively. Hypothalamic mRNA levels of all these genes were significantly (P < 0.005) increased at 4, 6, and 24h CLP, as compared to sham-operated animals. IL-1ra pre-treatment in these CLP animals significantly decreased IL-1 gene expression at all time points and also of iNOS, caspase-3, and HIF-1α at 24h when compared to vehicle-treated CLP animals. The effect of the pre-treatment on protein expression was most clearly seen for IL-1β and iNOS at 24h. Our results showed that blocking the IL-1-IL-1r signaling pathway by central administration of an IL-1ra decreases hypothalamic oxidative stress markers during sepsis.

Thrombin generation in abdominal sepsis is Rho-kinase-dependent

Sepsis causes severe derangements of the coagulation system. However, the signaling mechanisms regulating sepsis-induced thrombin generation remain elusive. Herein, we hypothesized that Rho-kinase might be an important regulator of thrombin generation in abdominal sepsis. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. Thrombin generation, coagulation factors, lung histology and myeloperoxidase (MPO) activity were determined 6 h and 24 h after induction of CLP. Induction of CLP triggered a systemic inflammatory response characterized by neutrophil accumulation and tissue injury in the lung as well as thrombocytopenia and leukocytopenia. Administration of Y-27632, a Rho-kinase inhibitor, attenuated these markers of systemic inflammation in CLP animals. Moreover, peak thrombin formation was decreased by 77% and 81% in plasma from mice 6 h and 24 h after induction of CLP. Total thrombin generation was reduced by 64% and 67% 6 h and 24 h after CLP induction, respectively. Notably, administration of Y-27632 increased peak formation by 99% and total thrombin generation by 66% in plasma from septic animals. In addition, CLP markedly decreased plasma levels of prothrombin, factor V and factor X at 6 h and 24 h. Interestingly, Rho-kinase inhibition significantly enhanced levels of prothrombin, factor V and factor X in plasma from septic mice. In addition, inhibition of Rho-kinase decreased CLP-induced elevations of CXCL2 by 36% and interleukin-6 by 38%. These novel findings suggest that sepsis-induced thrombin generation is regulated by Rho-kinase. Moreover, inhibition of Rho-kinase reverses sepsis-evoked consumption of coagulation factors. Thus, our results show that targeting Rho-kinase signaling might protect against coagulation dysfunction in abdominal sepsis.