Dehydroepiandrosterone: a modulator of cellular immunity and heat shock protein 70 production during polymicrobial sepsis

Objective DHEA is an immunomodulatory steroid hormone that improves survival during systemic inflammation. A DHEA-induced modulation of heat shock protein response may be an alternative mechanism contributing to the beneficial effects of this hormone. We investigated the effect of DHEA administration on survival, cellular immune functions, and HSP-70 production in septic mice. Design and setting Randomized animal study, level I trauma center, university research laboratory. Subjects Male NMRI mice. Interventions Mice were subjected to sham operation (laparotomy, LAP) or sepsis (cecal ligation and puncture, CLP) with or without administration of either saline 0.9% (LAP, CLP) or 20 mg/kg DHEA subcutaneously (LAP/DHEA, CLP/DHEA). Survival was monitored over a 48-h period. Splenocyte apoptosis rate (AnnexinV binding), splenocyte proliferation ([³H]thymidine incorporation), TNF-α plasma concentration (ELISA), and HSP-70 concentration (ELISA) in tissue extracts from liver, lung, and spleen were monitored 48 h after onset of sepsis. Results DHEA administration improved the survival of septic mice (78% vs. 50%). This effect was paralleled by increased splenocyte proliferation, decreased cellular apoptosis rate of splenocytes, and attenuation of TNF-α release. Furthermore, an increased HSP-70 concentration was observed in lungs and spleens of DHEA-treated septic animals. Conclusions DHEA-treatment decreased the mortality rate of septic mice. This was accompanied by improved cellular immune functions and an augmented heat shock response (HSP-70) of lungs and spleens. Further studies are required to demonstrate a direct relationship between the improved survival and the observed alterations in the immune system in DHEA-treated animals.     

Dehydroepiandrosterone modulates toll-like receptor expression on splenic macrophages of mice after severe polymicrobial sepsis

Toll-like receptors (TLRs) play a pivotal role in the induction of innate immunity after the transactivation of proinflammatory cytokine genes. However, the responses of TLRs during severe polymicrobial sepsis have not been thoroughly examined. Although dehydroepiandrosterone (DHEA), a steroid hormone, is reported to have an immunomodulatory effect after sepsis, the mechanism responsible for its salutary is not known. To investigate this, male ICR/Jcl mice (5-8 weeks old) were subjected to sepsis by cecal ligation and puncture (CLP) or sham operation. The mice received vehicle or DHEA (40 mg/kg body weight) subcutaneously immediately after the surgery. Plasma IL-10 levels and splenic macrophage TNF-alpha production, as well as the expression levels of CD14, TLR2, and TLR4 mRNAs on splenic macrophages, were assessed 6 h after the surgery. The results indicate that mice with sepsis show a marked increase in the plasma IL-10 levels and a decrease in TNF-alpha production by splenic macrophages. TLR2 and TLR4 mRNA expression levels after CLP were significantly lower compared with those after the sham operation. TNF-alpha production and TLR2 and TLR4 mRNA expression on splenic macrophages are restored with DHEA administration. Furthermore, administration of DHEA after CLP delayed the mortality of animals. These results indicate that the anti-inflammatory phase of sepsis induces a marked down-regulation of TLR expression on splenic macrophages; however, administration of DHEA resulted in the restoration of TLR2 and TLR4 mRNA expression.     

Cannabinoid antagonist AM 281 reduces mortality rate and neurologic dysfunction after cecal ligation and puncture in rats

OBJECTIVES: The purpose of this study was to examine whether anandamide, an endogenous cannabinoid receptor ligand, is involved in the pathogenesis of septic encephalopathy. DESIGN: Prospective, controlled study. SUBJECTS: Male Wistar rats (7 wks old) were randomly divided into four groups as follows: group 1, control (0.5 mL of saline injected subcutaneously); group 2, sham (surgical abdominal incision and suturing were performed, but ligation and puncture of the cecum were omitted); group 3, cecal ligation and puncture (CLP); group 4, CLP + AM 281 ([N-morpholin-4-yl]-5-[2,4-yl]-5-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide) as the cannabinoid receptor antagonist (1 mg/kg intraperitoneally). INTERVENTIONS: Sepsis was induced by CLP under pentobarbital anesthesia (10 mg/kg intraperitoneally) with 1% isoflurane. A 2-Fr high-fidelity micromanometer catheter was inserted into the left ventricle via the right carotid artery to assess hemodynamics. Each of the rats was neurologically assessed at 30 mins and 12, 24, and 48 hrs after the treatment. The cytoplasmic levels of caspase-3 in the hippocampi were assayed before surgery and at 30 mins and 24 and 48 hrs after surgery using Western blotting techniques. To examine the effects of AM 281 on neurologic function and mortality rate, we set another control group treated solely with AM 281. Selective inducible nitric oxide synthase inhibitor, L-N6-(1-iminoethyl)-lysine (4 mg/kg), was injected intraperitoneally immediately after CLP to produce the CLP + L-N6-(1-iminoethyl)-lysine group to exclude the influence of depressed hemodynamics on neurologic impairment. MEASUREMENTS AND MAIN RESULTS: It was found that administration of AM 281 could prevent the hemodynamic changes induced by sepsis. Reflex responses, including the pinna, corneal, paw or tail flexion, and righting reflexes, and the escape response significantly decreased in the CLP and CLP + L-N6-(1-iminoethyl)-lysine groups at 48 hrs after the surgery. In contrast, no changes in these reflex responses were found between the CLP + AM 281 and control and sham groups. In addition, no effects of the administration of AM 281 on neurologic function and mortality rate in the control group were found. Tissue caspase-3 levels were elevated at 48 hrs after CLP in the CLP alone group (means +/- sd: control, 3.9 +/- 0.4; sham, 4.2 +/- 0.4; CLP, 7.1 +/- 1.0 [p < .01]; CLP + AM 281, 4.0 +/- 0.5 densitometric units). In addition, administration of AM 281 also decreased the mortality rate (p < .05). CONCLUSIONS: Administration of AM 281 prevented the hemodynamic changes and development of neurologic dysfunction occurring in association with septic shock, and could decrease the mortality rate in experimentally induced septic shock in rats. Although further studies are necessary to determine whether endogenous cannabinoids cause septic encephalopathy in rats directly or via their effects on systemic hemodynamics, the beneficial effects of AM 281 on these rats might have significant therapeutic implications in cases of septic encephalopathy.     

Dehydroepiandrosterone (DHEA) modulates the activity and the expression of lymphocyte subpopulations induced by cecal ligation and puncture

Dehydroepiandrosterone (DHEA) exerts a variety of positive effects on the immunologic alterations after trauma and sepsis. We therefore measured the therapeutic efficacy of DHEA after cecal ligation and puncture (CLP) on the expression of lymphocyte subpopulations and on the delayed type hypersensitivity (DTH) reaction. Male NMRI-mice were randomly assigned to four different treatment groups. Treatment consisted of DHEA or saline (S) administration after CLP or laparotomy only. Flow cytometry was performed (CD4+, CD8+, and CD56 lymphocytes) after 96 hours. DTH-reaction, activity and mortality rate were documented. The CLP-induced reduction in activity and survival (mortality: 34/40) was significantly (p < 0.03) less sustained in CLP-DHEA (mortality: 22/40). The DTH-ratio (before vs. after secondary challenge) was significantly lowered in CLP-S (1.01 +/- 0.15) compared to CLP-DHEA (1.35 +/- 0.1) after 48 hours (p < 0.01). CLP-DHEA (22.2 +/- 7.9%) was associated with a statistically significant less sustained increase of CD56+ cells (p < 0.01) compared with CLP-S (49.0 +/- 6.9%). DHEA-treatment after CLP was associated with less reduction in the CD8+ T-lymphocyte subsets (p < 0.01 vs. all other groups). DHEA treatment after CLP was associated with fewer alterations in the changes of CD8+ and CD56, cells, and the DTH reaction compared with animals submitted to CLP without any treatment. This difference was associated with improved outcome (reactivity, mortality). These results suggest a modulation at specific immune reactions by DHEA treatment.     

Sepsis-induced lung injury in rats increases alveolar epithelial vulnerability to stretch

OBJECTIVE: Previous in vitro models have shown that cellular deformation causes dose-dependent injury and death in healthy rat alveolar epithelial cells (AECs). We compared the viability of AECs from septic rats with those from nonseptic rats after 1 hr of cyclic equibiaxial stretch. We hypothesized that sepsis would increase stretch-induced cell death. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Thirty-seven male Sprague-Dawley rats weighing 240-260 g. INTERVENTIONS: Anesthetized rats were subjected to cecal ligation and double puncture (2CLP) or sham laparotomy without cecal ligation or puncture (sham). After 24 or 48 hrs, AECs were isolated, seeded in custom wells, and maintained in culture for 48 hrs before study. AECs were stretched cyclically (15/min) to a 0%, 12%, 25%, or 37% change in surface area (DeltaSA) for 1 hr. Cell viability, phenotypic markers, and nuclear factor-kappaB intracellular localization were assessed using fluorescent immunocytochemistry. MEASUREMENTS AND MAIN RESULTS: Phase and fluorescent images were evaluated for all studies. Response to stretch was the same at 24 and 48 hrs after 2CLP. Relative to sham, 2CLP significantly increased cell death at 25 and 37% DeltaSA (p<.003, analysis of variance). Relative to sham, 2CLP did not alter expression of type I or type II phenotypic markers. Nuclear factor-kappaB within the nuclear compartment was observed after 2CLP in unstretched cells and after 1 hr of cyclic stretch at 37% DeltaSA. In sham, nuclear factor-kappaB within the nuclear compartment was seen only after stretch. CONCLUSIONS: AECs isolated from septic rats are more vulnerable to mechanical deformation injury than AECs from nonseptic animals.     

The anti-inflammatory effect of curcumin in an experimental model of sepsis is mediated by up-regulation of peroxisome proliferator-activated receptor-gamma

OBJECTIVE: Although phytochemical curcumin has been shown to possess anti-inflammatory properties, it remains unknown whether this agent has any beneficial effects in sepsis. The purpose of this study was to demonstrate whether curcumin protects septic animals and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR)-gamma, an anti-inflammatory nuclear receptor, plays any role. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: A bolus injection of 0.2 micromol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 micromol/day) for 3 days via a primed 2-mL mini-pump. The rats were then subjected to sepsis by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS: Serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase), lactate, albumin, and tumor necrosis factor (TNF)-alpha were measured at 20 hrs after CLP (i.e., late stage of sepsis). In addition, a 10-day survival curve was conducted following CLP and cecal excision with or without curcumin treatment. Furthermore, macrophages cell line RAW 264.7 cells were treated with curcumin followed by stimulation with endotoxin. TNF-alpha and PPAR-gamma expression were then measured. The results indicate that intravenous administration of curcumin before the onset of sepsis attenuated tissue injury, reduced mortality, and decreased the expression of TNF-alpha in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-gamma in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-gamma antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-alpha expression and markedly up-regulated PPAR-gamma expression without affecting cell viability. Curcumin also prevented morphologic alterations in macrophages induced by endotoxin. CONCLUSIONS: The protective effect of curcumin makes it or its analogues strong candidates as a novel therapy for sepsis. The beneficial effect of curcumin appears to be mediated by up-regulation of nuclear receptor PPAR-gamma.     

Dopamine affects cellular immune functions during polymicrobial sepsis

Objective To determine whether infusion of dopamine modulates cellular immune functions and survival during systemic inflammation. Design and setting Randomized animal study, university research laboratory, Level I trauma center. Subjects Male NMRI mice. Interventions Mice were subjected to laparotomy (sham intervention, LAP) or polymicrobial sepsis induced by cecal ligation and puncture (CLP). Mice in each of these conditions received either an intraperitoneal infusion of 0.9% saline (CLP/saline; LAP/saline) or an intraperitoneal infusion of dopamine (1.0 μg/kg/min i.p., CLP/DOP; LAP/DOP). Metabolic data and survival were monitored 24 h and 48 h after onset of sepsis, and animals were terminated 48 h after induction of sepsis to determine splenocyte apoptosis (Annexin V binding capacity), splenocyte proliferation (³H-Thymidine incorporation assay), splenocyte IL-2, IL-6 and IFN-γ release (ELISA) and leukocyte distribution (WBC; CD3, CD4, CD8, B220, F4/80, NK1.1). Measurements and results Infusion of dopamine in septic mice increased splenocyte apoptosis and decreased splenocyte proliferation and IL-2 release of septic mice. Furthermore, an inhibitory effect of dopamine infusion on splenocyte proliferation and the release of the TH1-cytokines IL-2 and IFN-γ was observed in sham operated control mice. These effects were paralleled by a decreased survival of dopamine-treated septic animals (47% vs. 67%). Treatment with DOP did not affect sepsis-induced changes of leukocyte distribution. Conclusions We conclude that dopamine is capable of modulating cellular immune functions in a murine model of sepsis. Michael S. Exton is currently affiliated with Eli Lilly Australia Pty LtdThis article is discussed in the editorial available at:     

Protective effects of penehyclidine hydrochloride on septic mice and its mechanism

Anticholinergics can have protective effects against septic shock. Penehyclidine hydrochloride (PHC) is a novel anticholinergic agent exhibiting few cardiovascular side effects. This work explored the protective effects of PHC on septic mice and its mechanism. Mice were randomly divided into four groups: sham control, cecal ligation and puncture (CLP), CLP/0.3 mg/kg PHC, and CLP/0.45 mg/kg PHC, with 10 mice in each. One hour before surgery, PHC-treated mice received an intraperitoneal injection of PHC and an equal volume of saline in the other two groups. Blood plasma and tissue samples were collected at 12 h after surgery. Serum TNF-alpha, histopathology, superoxide dismutase (SOD), malondialdehyde (MDA), and expression of iNOS in lung and hepatic tissues were examined. Another 40 mice were randomly assigned to four equal groups to observe survival status during 96 h after operation. Treatment of 0.45 mg/kg PHC markedly decreased TNF-alpha, MDA content, and iNOS mRNA expression, and enhanced SOD activity (P < 0.05 and P < 0.01). Treatment of 0.45 mg/kg PHC might have a protective effect against sepsis. Its action mechanisms are probably involved in the inhibition of inflammatory factor production and suppression of iNOS mRNA expression and lipidperoxidation.     

Delayed ascorbate bolus protects against maldistribution of microvascular blood flow in septic rat skeletal muscle

OBJECTIVE: Although early administration of ascorbate has been shown to protect against the microvascular dysfunction in sepsis, it is not clear if a delayed introduction of ascorbate also yields beneficial effects. The main objective was to determine the therapeutic window for treatment of an animal model of sepsis with bolus injection of ascorbate. We also determined if sepsis per se affects urinary excretion of ascorbate. DESIGN: Prospective, controlled laboratory study. SETTING: Animal laboratory in a university-affiliated research institute. SUBJECTS: Male Sprague-Dawley rats, 300-400 g of body weight. INTERVENTIONS: Rats were made septic by cecal ligation and perforation (CLP) and volume resuscitated by continuous saline infusion. Ascorbate bolus (7.6 mg/100 g of body weight) or saline vehicle was injected intravenously at 1, 6, or 24 hrs after CLP. MEASUREMENTS AND MAIN RESULTS: At 24 hrs post-CLP, sepsis caused antidiuresis and decreased plasma ascorbate concentration, but it did not affect urinary excretion of ascorbate in rats that received only saline. Sepsis also caused maldistribution of capillary blood flow in skeletal muscle. This maldistribution of flow was prevented by ascorbate injected at 6 hrs post-CLP. At 48 hrs post-CLP, in addition to the flow maldistribution, sepsis caused systemic arterial hypotension and fever that were prevented by both immediate (1 hr post-CLP) and delayed injections of ascorbate (24 hrs post-CLP). CONCLUSION: Despite volume resuscitation, the present model of sepsis resulted in maldistribution of capillary blood flow within 24 hrs and hypotension within 48 hrs. Our finding that intravenous bolus of ascorbate can protect against these deficits even if delayed 6-24 hrs after the septic insult shows, for the first time, that ascorbate can reverse microcirculatory dysfunction after the onset of sepsis.     

丙酮酸乙酯对脓毒症大鼠肠黏膜屏障功能保护作用的研究

目的 观察丙酮酸乙酯(ethyl pyruvate ,EP)干预治疗对脓毒症大鼠生存率和肠黏膜屏障的影响.方法 ①EP对脓毒症大鼠生存率的影响:无特定病原雄性SD大鼠100只随机分为假手术组(A组)、脓毒症组(B组)、EP早期治疗组(C组)及EP延迟治疗组(D组),每组25只,利用盲肠结扎穿孔法(cecal ligation and puncture,CLP)制作大鼠脓毒症模型,各组均于术后6、12、18、24、36、48、60、72 h腹腔内注射给药3 mL,C、D组分别于术后6、12 h开始予EP(40 mg/kg),A、B两组同法予等量林格乳酸钠溶液(ringer lactate solution ,RLS),每隔12 h记录死亡情况,分析比较5 d生存率;②EP对脓毒症大鼠肠黏膜屏障的影响:80只无特定病原雄性SD大鼠随机分为四组,每组20只,分组及给药方法与方法一相同,术后24、48 h各处死10只.测定各时间点血浆D-乳酸、DAO的变化,同时用透射电镜观察术后48 h肠黏膜上皮细胞超微结构的变化.采用Kaplan- Meier生存分析法进行生存分析,多组均数间比较采用单因素方差分析的方法, 多组均数间两两比较采用SNK-q检验,P＜0.05为差异有统计学意义.结果 A、B、C、D四组大鼠5 d生存率分别为100%、24%、68%、56%,与B组相比,C、D组大鼠5 d生存率明显提高(P<0.05),C、D组间差异无统计学意义(P＞0.05);与B组相比,C、D组术后24 h和48 h血浆D-乳酸含量明显下降(P<0.01);与B组相比,C组、D术后24 h和48 h血浆DAO活性明显下降(P<0.01),C、D组术后24、48 h血浆D-乳酸含量、DAO活性差异无统计学意义(P＞0.05);电镜下C、D组肠黏膜上皮细胞损伤较B组明显减轻,细胞间紧密连接较清楚.结论 脓毒症时肠黏膜损伤严重,EP早期与延迟干预治疗能有效保护肠黏膜屏障,提高5 d生存率,具有抗脓毒症作用 .     

High-dose exogenous iron following cecal ligation and puncture increases mortality rate in mice and is associated with an increase in gut epithelial and splenic apoptosis

OBJECTIVES: Despite having dysregulated iron metabolism, critically ill patients may receive exogenous iron for the treatment of anemia. Iron is associated with increased tissue apoptosis and may facilitate bacterial growth. We hypothesized that exogenous iron administration given after the onset of sepsis would lead to increased mortality rate. To discriminate between elevated cell death and bacterial overgrowth as potential mediators of mortality, we examined gut epithelial and lymphocyte apoptosis and systemic bacterial counts in animals given iron supplementation after the onset of sepsis. DESIGN: Prospective, randomized, controlled study. SETTING: Animal laboratory in a university medical center. SUBJECTS: Male C57BL/6 mice, 6-10 wks old. INTERVENTIONS: C57BL/6 mice were subjected to cecal ligation and puncture (CLP), a well-accepted model of intra-abdominal sepsis, followed by daily subcutaneous injections of either 1 mL of iron dextran (5 mg/mL) or 0.9% NaCl for a total of five doses. Animals (n = 78) were followed for survival for 8 days. Separate cohorts (n = 76) were killed 24 or 48 hrs after cecal ligation and puncture or sham laparotomy and were assayed for gut epithelial and splenic apoptosis as well as for quantitative blood cultures. MEASUREMENTS AND MAIN RESULTS: Eight-day survival was 7% in animals that received iron and 26% in mice that received 0.9% NaCl (p < .005). Iron supplementation after cecal ligation and puncture increased apoptosis by both active caspase 3 and hematoxylin and eosin staining in both the intestinal epithelium and spleen at 24 hrs (p < .05). Iron supplementation after sham laparotomy did not cause mortality or elevated apoptosis. Quantitative blood cultures revealed no detectable differences between septic animals that received iron and those that received 0.9% NaCl. CONCLUSIONS: High-dose iron supplementation with iron dextran after the onset of sepsis significantly increases mortality rate in this animal model. Iron-induced mortality may be mediated by an increase in gut epithelial and splenic apoptosis, whereas severity of bacteremia does not appear to play a causative role.     

The activity of tissue factor pathway inhibitor in experimental models of superantigen-induced shock and polymicrobial intra-abdominal sepsis

OBJECTIVES: To study recombinant human tissue factor pathway inhibitor (rhTFPI) in a superantigen-induced shock model and in a cecal ligation and puncture (CLP) model of peritonitis in mice. DESIGN: Prospective, randomized, experimental study. SETTING: An experimental animal research laboratory. SUBJECTS: Eighty BALB/c mice for the superantigen model, and 56 BALB/c mice for the CLP model. INTERVENTIONS: In the superantigen-induced shock model, animals received rhTFPI (350 mg/kg) subcutaneously every 12 hrs (n = 30) or saline control (n = 30) for 60 hrs after staphylococcal enterotoxin B (SEB; 10 microg iv) and a sublethal dose of E. coli 0111:B4 lipopolysaccharide (LPS; 75 microg ip). Control groups received SEB alone (n = 10) and LPS alone (n = 10). In the CLP model, rhTFPI or saline was given every 8 hrs for 48 hrs by using a 21-gauge needle (n = 9) or 23-gauge needle (n = 14) for CLP. A sham surgery control group (n = 10) was also included. MEASUREMENTS AND MAIN RESULTS: There was 0% mortality in the SEB and LPS control groups. The mortality rate was 64% in the saline control group that received both SEB and LPS (19 of 30), whereas the rhTFPI- treated animals had a mortality rate of 20% (6 of 30; p < .01). The rhTFPI-treated group had significantly lower interleukin-6 levels (61.8 +/- 41 pg/mL vs. 285 +/- 63 pg/mL; p < .05) than the control group but no differences in tumor necrosis factor-alpha or interferon-gamma levels. In the CLP experiment, rhTFPI-treated animals did not have any survival advantage over the control group after the large-bore (21-gauge) needle puncture. The rhTFPI group had significantly improved 7-day mortality rate after CLP with the small-bore needle (23-gauge; 21.4% [rhTFPI] vs. 71.4% [control], p < .01). Plasma LPS, interleukin-6, interferon-gamma, and tumor necrosis factor-alpha levels were unchanged by rhTFPI treatment, but significantly reduced LPS (p = .006) and IFNgamma (p = .001) levels were found in the peritoneal fluid. CONCLUSIONS: Tissue factor pathway inhibitor significantly improves the mortality rate in models of superantigen-induced shock and polymicrobial intra-abdominal infection, supporting its potential use in clinical trials for septic shock.     

Hyperbaric oxygen protects from sepsis mortality via an interleukin-10-dependent mechanism

OBJECTIVE: This study was performed to determine whether hyperbaric oxygen (HBO2) therapy is protective in cecal ligation and puncture (CLP)-induced sepsis and if protection is dependent on oxygen dosing. We also wished to determine whether HBO2 affected bacterial clearance or altered macrophage production of interleukin-10 (IL-10)s in the setting of CLP sepsis. Finally, we wished to determine whether the mechanism of HBO2 protection in sepsis was dependent on IL-10 production. DESIGN: Prospective, experimental study. SETTING: University experimental research laboratory. SUBJECTS: C57BL/6 and C57BL/6 IL-10 mice. INTERVENTIONS: Sepsis was induced by CLP. Mice were randomized to receive a 1.5-hr HBO2 treatment at either 1, 2.5, or 3 atmospheres absolute every 12 hrs or HBO2 at 2.5 atmospheres absolute every 24 hrs. Mice were also harvested at 24 hrs for determination of bacterial load and isolation and study of CD11b peritoneal macrophages. MEASUREMENTS AND MAIN RESULTS: Survival was monitored for 100 hrs after CLP +/- HBO2 treatment. HBO2 significantly improved survival when administered at 2.5 atmospheres absolute every 12 hrs. Other treatment schedules were not protective, and treatment at 3.0 atmospheres absolute significantly worsened survival outcome. Bacterial load was significantly reduced in splenic homogenates but not peritoneal fluid at 24 hrs. Macrophages isolated from HBO2-treated mice demonstrated enhanced IL-10 secretion in response to lipopolysaccharide as compared with CLP controls. Mice genetically deficient in IL-10 expression treated with HBO2 at 2.5 atmospheres absolute every 12 hrs were not protected from CLP-induced mortality. CONCLUSION: HBO2 may be protective in CLP sepsis within a window of oxygen dosing. The mechanism of HBO2 protection may be potentially linked in part to expression of IL-10, as peritoneal macrophages demonstrated enhanced IL-10 expression and IL-10 mice were not protected by HBO2 treatment.     

Adrenal insufficiency during the late stage of polymicrobial sepsis

OBJECTIVES: Although studies have indicated that adrenal insufficiency occurs after severe trauma and hemorrhagic shock, it remains controversial whether adrenal function is depressed during the late stage of polymicrobial sepsis. DESIGN: Prospective, controlled animal study. SETTING: A university research laboratory. SUBJECTS: Male rats (275-325 g) were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP) or sham operation followed by the administration of normal saline solution. MEASUREMENTS AND MAIN RESULTS: Systemic blood samples were taken at 20 hrs after CLP (i.e., a late stage of sepsis) or sham operation to measure plasma levels of corticosterone and corticotropin as well as adrenal contents of corticosterone. Additional groups of animals were utilized to examine corticotropin-stimulated plasma corticosterone release as well as adrenal levels of cyclic adenosine monophosphate (cAMP, the second messenger of corticotropin action). The results indicate that despite a 75% (p < .05) higher concentration in plasma corticotropin at 20 hrs after the onset of sepsis, plasma corticosterone levels were similar to those in sham-operated animals. In addition, adrenal contents of corticosterone were reduced by 42% (p < .05) in septic animals. Moreover, the plasma corticosterone and adrenal cAMP responses to corticotropin were reduced by 53% and 27% (p < .05), respectively, at 20 hrs after CLP. CONCLUSIONS: These findings suggest that, despite high plasma levels of endogenous corticotropin, adrenal dysfunction, as indicated by the reduction of corticotropin-induced plasma corticosterone release and adrenal contents of cAMP as well as the decreased adrenal levels of corticosterone, occurs during the late stage of polymicrobial sepsis. Therefore, the recognition of adrenal insufficiency and interventions to improve adrenal responsiveness may be beneficial in improving the outcome during late sepsis.     

The importance of systemic cytokines in the pathogenesis of polymicrobial sepsis and dehydroepiandrosterone treatment in a rodent model

The pathogenesis of sepsis is still undetermined to a large extent. It is an established fact that female gender is associated with a lower mortality and that sex steroid hormones influence the immunologic response. Dehydroepiandrosterone (DHEA) seems to have a protective immunologic effect in sepsis. It is still unknown in which way DHEA influences the pathogenesis of sepsis. Therefore, the effect of DHEA application on cytokine concentrations in tumor necrosis factor (TNF) receptor (TNF-RI(-/-)) and interleukin-6 (IL-6(-/-)) knockout mice was determined. In a model of polymicrobial sepsis induced by coecal ligation and puncture (CLP), the effect of DHEA on survival and cytokine concentrations was examined. For clarification of the role of TNF-RI, CLP was performed in TNF-RI knockout mice (TNF-RI(-/-)). In addition, IL-6 knockout mice (IL-6(-/-)) were used to clarify the role of IL-6. Furthermore, experiments were performed in mice that were not genetically modified (wild type, WT). The protective effect of DHEA could be confirmed in this CLP model. DHEA application was associated with a reduction in mortality in WT animals. Moreover, DHEA-treated animals demonstrated a reduction in systemic inflammatory effects, as determined by proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and the antiinflammatory cytokine IL-10. In this work, it was shown that the TNF-RI is essential for survival after CLP. DHEA application was associated with a reduction of mortality of 100% in TNF-RI(-/-) mice after CLP to 50%. This result engages, that the effect of DHEA is TNF-RI independent. However, the application of DHEA had no influence on the mortality in IL-6-/- mice. It can be concluded that the protective effect of DHEA in polymicrobial sepsis is mediated IL-6 dependently. DHEA reduces the systemic inflammation, measurable via the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and the antiinflammatory cytokine IL-10. IL-6 might be involved in the DHEA-mediated reduction of postseptic complications. In contrast, DHEA seems to be TNF-RI independent. Consequently, DHEA might be useful as an adjunct therapy for the immune modulation in sepsis.     

Treatment with N-acetylcysteine plus deferoxamine protects rats against oxidative stress and improves survival in sepsis

OBJECTIVE: Oxidative stress plays an important role in the development of multiple organ failure and septic shock. Here we have evaluated the effects of a combination of antioxidants (N-acetylcysteine plus deferoxamine) in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). DESIGN: Prospective, randomized, controlled experiment. SETTING: Animal basic science laboratory. SUBJECTS: Male Wistar rats, weighing 300-350 g. INTERVENTIONS: Rats subjected to CLP were treated with either N-acetylcysteine (20 mg/kg, 3 hrs, 6 hrs, 12 hrs, 18 hrs, and 24 hrs after CLP, subcutaneously) plus deferoxamine (20 mg/kg, 3 hrs and 24 hrs after CLP, subcutaneously) or vehicle with or without "basic support" (saline at 50 mL/kg immediately and 12 hrs after CLP plus ceftriaxone at 30 mg/kg and clindamycin 25 mg/kg every 6 hrs). MEASUREMENTS AND MAIN RESULTS: After 12 hrs, tissue myeloperoxidase (indicator of neutrophil infiltration), thiobarbituric acid reactive species (as a marker of oxidative stress), catalase and superoxide dismutase activities (antioxidant enzymes), and mitochondrial superoxide production (index of uncoupling of electron transfer chain) were measured in major organs involved in septic response. Rats treated with antioxidants had significantly lower myeloperoxidase activity and thiobarbituric acid reactive species formation in all organs studied. Mitochondrial superoxide production was significantly reduced by antioxidant treatment. Furthermore, antioxidants significantly improved the balance between catalase and superoxide dismutase activities. Survival in untreated septic rats was 10%. Survival increased to 40% with fluids and antibiotics. In rats treated only with N-acetylcysteine plus deferoxamine, survival was also significantly improved (47%) in a manner similar to basic support. Survival increased to 66% with basic support with N-acetylcysteine plus deferoxamine. CONCLUSIONS: Our data provide the first experimental demonstration that N-acetylcysteine plus deferoxamine reduces the consequences of septic shock induced by CLP in the rat, by decreasing oxidative stress and limiting neutrophil infiltration and mitochondrial dysfunction, thereby improving survival.