Response In Septic Rats Research Articles

Effect of CD4+ CD25+ regulatory T cells and T helper type 17 cells in septic rats

Objective To investigate the roles of CD4+ CD25+ regulatory T cells(Treg)and T helper type 17(Th17)cells in inflammatory response in septic rats. Methods A total of 110 healthy male Sprague-Dawley rats(250-300 g)were randomly divided into 3 groups:normal control group,sham group and cecal ligation and puncture(CLP)group.The rat models of sepsis were established by improved CLP.Flow cytometric analysis was performed to detect the percentage of CD14+ T cell, Th17 cells and Treg cells subpopulation.Expressions of proinflammatory cytokines[interleukin(IL)-6,IL-10,tumor necrosis factor(TNF)-α, transforming growth factor(TGF)-β, IL-17] were measured by using enzyme-linked immunosorbent serologic assay. Results Compared with sham group,(1) While the spesis becoming worse, the rats were immunological inhibition, the expression levels of leucocyte antigen-DR(19. 63±7. 56)% lower than 30% and the rate of IL-10/TNF-α(27. 41±7. 04 vs.6. 63±2. 60, P< 0. 01)significantly up-regulated.(2) After 96 hours, the proportions of Treg cells[(11. 91±3. 88)% vs.(6. 57±2. 60)%, P< 0. 01]and Th17 cells[(5. 14±0. 29)% vs.(2. 85±0. 07)%,P<0. 01]in sepsis group were significantly higher.(3)After 96 hours, the expression levels of IL-6[(42. 31±15. 89) ng/L vs.(6. 32±3. 18) ng/L, P< 0. 01], IL-10 [(69. 89±20. 78)ng/L vs.(13. 58±5. 37) ng/L, P< 0. 01], TNF-α[(5. 03±3. 10) ng/L vs.(2. 77± 1. 10)ng/L, P<0. 01], TGF-β[(4. 99±2. 01)ng/L vs.(1. 88±1. 07)ng/L,P<0. 01],IL-17[(92. 77 ±11. 64)ng/L vs.(7. 58±2. 30)ng/L, P<0. 01]were significantly up-regulated in sepsis group. Conclusion While the spesis becoming worse, the rats were immunological inhibition.Aberrant activation of Th17 cells and Treg cells might be involved in pathogenesis in sepsis.The changes of cytokine environment in sepsis may be one of the factors causing the imbalance of Treg cells/Th17 cells. Key words: CD4+ CD25+ regulatory T cells; T helper type 17 cells; Sepsis

Protective effect of erythropoietin against myocardial injury in rats with sepsis and its underlying mechanisms.

The aim of this study was to investigate the protective effect of erythropoietin (EPO) against acute myocardial injury and its underlying mechanisms. Mice (n=146) were randomly divided in a double-blind manner into four groups, sham, Rocephin, EPO and sepsis, and mortality was observed on the seventh day after cecal ligation and puncture. In addition, a total of 252 rats were randomly divided into three groups, sham, EPO and sepsis, and indicators of cardiac function, inflammatory mediators and serum creatine kinase levels were assessed. Mitochondrial membrane potential, cell apoptosis and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) p65 expression levels were detected using flow cytometry. Following intervention with EPO, the mortality rate in mice with sepsis was significantly reduced and the cardiac function of septic rats was significantly improved. In addition, the levels of inflammatory mediators, serum creatine kinase and apoptosis and the myocardial mitochondrial membrane potential and expression of NF-κB p65 in cardiac tissue were all improved following EPO treatment, and the differences between the results for the sepsis and EPO groups were statistically significant (P<0.05). These findings suggest that EPO reduces the myocardial inflammatory response in septic rats, attenuates the reduction in mitochondrial membrane potential and inhibits myocardial cell apoptosis by reducing NF-κB p65 expression, and therefore exerts a protective effect in the myocardium.

Dose-Related Effects of Hyperoxia on the Lung Inflammatory Response in Septic Rats

We evaluated the effects of hyperoxia on pulmonary inflammatory changes in sepsis induced by cecal ligation and puncture (CLP) in rats. Seven groups were studied: sham-operated rats breathing air for 20 or 48 h; CLP breathing air for 20 or 48 h; and CLP + 100% oxygen for 20 h, or 70% oxygen for 48 h, or 100% oxygen intermittently (6 h/d) for 48 h. Video microscopy was used to monitor lung macromolecular leak, microvascular flow velocity, and shear rates, and lung morphometry was used for leukocyte infiltration and solid tissue area. Cell counts, tumor necrosis factor α, and nitrites were determined in peripheral blood and lung lavage fluid. Expression of adhesion molecules in blood leukocytes was evaluated by flow cytometry. Cecal ligation and puncture induced inflammation manifested in leukopenia, left shift, thrombocytopenia, increased expression of L selectin and CD11, increased serum and lavage fluid tumor necrosis factor α and leukocytes, and increased lung tissue area, macromolecular leak, and sequestration of leukocytes. Inhalation of 100% oxygen for 20 h increased nitrites (P < 0.01) and decreased leukocyte count in lavage fluid (P < 0.05) and attenuated lung macromolecular leak and changes in solid tissue area (P < 0.01). Inhalation of 70% oxygen (48 h) attenuated expression of adhesion molecules (P < 0.001) but failed to attenuate markers of lung inflammation. In contrast, intermittent 100% oxygen exerted favorable effects on markers of inflammation, attenuated leukocyte expression of L selectin and CD11 (P < 0.01), decreased pulmonary sequestration of leukocytes (P < 0.001), and ameliorated changes in macromolecular leak (P < 0.01) and lung solid tissue area (P < 0.05). Our data support the beneficial effects of safe subtoxic regimens of normobaric hyperoxia on the systemic and pulmonary inflammatory response following CLP.

The effects of immunosuppressive agents on inflammatory response in septic rats

Abstract Sepsis remains a serious clinical problems resulting in high morbidity and mortality. We aimed to investigate the inflammatory response in a septic rat model treated with immunomodulator agents. We used the cecal ligature and puncture model to establish septic peritonitis in rats. Male Wistar-Albino rats were randomized into groups of seven rats each and assigned different regimens: Tacrolimus 1 mg/kg/day, cyclosporin-A 5 mg/kg/day and methylprednisolone 15 mg/kg/day. These immunsuppressive agents were applied at the 6 and 48 hour intraperitoneally. The animals were euthanized after 6 and 48 hours and systemic parameters including, IL-2, IL-6, TNF-a, CRP, AST and creatinine were examined. Our study demonstrated that the experimental peritonitis model caused a meaningful rise in the values of systemic parameters. This was especially apparent for early-applied cyclosporin A; in addition, tacrolimus significantly decreased the levels both at the 6 and 48 hour. The excess immune response in complex sepsis treatment might be restrained using immunosuppressive agents administered early. Although additional supportive, comprehensive, experimental and clinical studies are still needed, this therapy model may prove to be an alternative for the future.

β-Adrenergic Blockade Exacerbates Sepsis-Induced Changes in Tumor Necrosis Factor α and Interleukin-6 in Skeletal Muscle and is Associated With Impaired Translation Initiation

Sepsis stimulates the sympathetic nervous system. The resultant elevation in plasma catecholamines, both norepinephrine and epinephrine (Epi), might be expected to alter the expression of inflammatory cytokines, which may directly or indirectly influence muscle protein balance. The purpose of this study was twofold: (1) determine whether Epi per se increases cytokine expression in skeletal muscle, and (2) determine whether beta-adrenergic blockade alters the sepsis-induced expression of inflammatory cytokines and mediators of protein balance in skeletal muscle. In the first study, rats were infused with Epi for 2 hour to increase the circulating Epi concentration to levels seen in septic animals. In the second study, sepsis was induced by cecal ligation and puncture and a nonspecific beta-adrenergic blockade produced with a continuous infusion of propranolol (PP). Tissues were obtained 24 after induction of sepsis and analyzed for tumor necrosis factor (TNF)-alpha interleukin (IL)-1beta, IL-6 mRNA and protein content. In addition, the tissue content of insulin-like growth factor (IGF)-I and various regulators of protein synthesis were assessed. Epi acutely increased TNF-alpha IL-6 and IL-1beta mRNA content in muscle (3- to 40-fold). However, only the TNF-alpha and IL-6 protein content was increased in muscle by Epi. In the second study, beta-adrenergic blockade with PP exacerbated the sepsis-induced increase in muscle IL-6 and TNF-alpha mRNA but did not alter the increment in IL-1beta or HMGB1. Propranolol also accentuated the sepsis-induced increase in both IL-6 and TNF-alpha protein in muscle. The exaggerated muscle cytokine response in septic rats treated with PP was associated with a reduction in muscle IGF-I protein that was greater than detected in saline-infused septic rats. Finally, the combination of sepsis + PP also accentuated the sepsis-induced decrease in the phosphorylation of 4E-binding protein-1, ribosomal protein S6, and mTOR, which are key proteins controlling protein synthesis. These results demonstrate that although Epi is capable of increasing tissue cytokines in naive rats, inhibition of the beta-adrenergic effects of catecholamines exacerbates the sepsis-induced increase of selected inflammatory cytokines. This exaggerated tissue response is associated with alterations in muscle IGF-I protein and translation initiation, which would be expected to impair tissue protein synthesis.

Effects of glutamine supplementation on innate immune response in rats with gut-derived sepsis

The present study examined the effect of glutamine (Gln)-enriched diets before sepsis or Gln-containing total parenteral nutrition (TPN) after sepsis, or both, on the phagocytic activity and blood lymphocyte subpopulation in rats with gut-derived sepsis. Rats were assigned to a control group or one of four experimental groups. The control group and groups 1 and 2 were fed a semipurified diet; groups 3 and 4 had part of casein replaced by Gln. After feeding the diets for 10 d, sepsis was induced by caecal ligation and puncture (CLP); TPN was maintained for 3 d after CLP. The control group and groups 1 and 3 were infused with conventional TPN and groups 2 and 4 were supplemented with Gln in the TPN solution. All rats were killed 3 d after CLP or sham operation to examine their immune responses. The results showed that compared with the control group, the phagocytic activities of peritoneal macrophages were enhanced in groups 3 and 4, but not in groups 1 and 2. The proportion of CD3+ cells in group 1 was significantly lower (P<0.05) than that of the control group, whereas no differences were observed among the control and Gln-supplemented groups. The CD4+ cell proportion was significantly lower (P<0.05) in group 1 compared with the control group and groups 3 and 4. These findings suggest that Gln-enriched diets before CLP significantly enhanced peritoneal macrophage phagocytic activity, preserved CD4+ cells and maintained blood total T lymphocytes in gut-derived sepsis. However, parenteral Gln administration after caecal ligation and puncture had no favourable effects on modulating immune response in septic rats.

Effects of the stress response in septic rats and LPS-stimulated alveolar macrophages: evidence for TNF-alpha posttranslational regulation.

We have previously demonstrated that induction of the stress response, by heat stress or sodium arsenite, administered 18 h before initiation of sepsis in rats, significantly decreased mortality and lung injury. As a possible mechanism underlying this effect, we hypothesized that the induction of the stress response, prior to bacterial endotoxin (lipopolysaccharide, LPS) stimulation, would cause a decrease in synthesis and/or release of tumor necrosis factor-alpha (TNF-alpha), making the animals more resistant to sepsis. Rats exposed to Salmonella typhosa LPS demonstrated a rise in plasma TNF-alpha. In contrast, rats exposed to heat stress or to sodium arsenite 18 h prior to LPS had significantly lower levels of plasma TNF-alpha. To examine the mechanisms by which the stress response mediates this decrease, we studied cultured alveolar macrophages. Similar to in vivo studies, TNF released into supernatants of alveolar macrophages treated with LPS was significantly higher than from cells exposed to the stress response prior to LPS. The decrease in TNF-alpha protein release was not accompanied by a similar decrease in TNF-alpha mRNA levels or by a decrease in cell-associated TNF-alpha, suggesting possible posttranslational regulation of TNF-alpha. To determine whether the decrease in TNF-alpha release was due to binding and sequestration by heat shock proteins (HSP), TNF-alpha was purified by immunoprecipitation. Under these conditions, TNF-alpha and HSP72kDa coprecipitated from cells that had received stress treatment prior to LPS. These data implicate HSP in posttranslational control of TNF-alpha release in LPS-stimulated alveolar macrophages exposed to the stress response.

Sustained hypermetabolic sepsis in rats: Characterization of the model

This paper describes the development of a sustained sepsis model, using chronically catheterized conscious unrestrained rats, which simulates the progression of septicemia in man, including a sustained hypermetabolic phase. Following chronic arterial catheterization, sepsis was induced in rats by intraperitoneal administration of 0.5 ml of a pooled fecal inoculum. The pooled inoculum, which was used to ensure a uniform inoculation of microorganisms to all animals, produced a septicemia which was progressively lethal. The resultant peritonitis was characterized as polymicrobial, with gram-negative bacteria being continuously present in both peritoneal fluid and blood. Septic animals were normotensive but tachycardic, compared to time-matched controls, throughout the observation period. In contrast to the stable colonic temperature of control animals, septic rats showed a significant febrile response on the first 3 days following inoculation. The hypermetabolic response in septic rats was also manifested by a 25, 38, and 28% increase in oxygen consumption on Days 1, 2, and 3 postinoculation. Animals responded to sepsis with a fall in blood glucose (on Day 2) which remained 15--20% below control levels. Mild hyperlactacidemia (2 mM) and reduced alanine concentrations (14--33%) were also seen in the septic group on Days 2 through 5. Despite the increased lactate levels, septic animals were mildly alkalotic (pH 7.51) which probably reflected the increased (32%) respiratory rate. Light microscopic findings in the septic animals revealed a spectrum of morphologic lesions including an extensive fibrinopurulent exudate, bacterial colonies, and abscesses, which involved most of the abdominal viscera. This investigation characterizes an experimental model of sustained sepsis in rats which exhibits hemodynamics, metabolic and pathologic alterations similar to those seen in human peritonitis.