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.     

Antibiotics delay but do not prevent bacteremia and lung injury in murine sepsis

OBJECTIVES: To investigate the effect of antibiotics on infection, lung injury, and mortality rate in polymicrobial sepsis and to determine whether an association exists between infection and lung injury and mortality rate. To circumvent the effect of antibiotics on cultures, we used polymerase chain reaction to detect bacteria. DESIGN: Prospective, randomized, controlled laboratory trial. SETTING: University research laboratory. SUBJECTS: C57/BL6 mice. INTERVENTIONS: Mice underwent cecal ligation and puncture without antibiotics (CLP) or with imipenem (CLP + Abx). MEASUREMENTS AND MAIN RESULTS: CLP resulted in 50% mortality rate at 48 hrs and 100% mortality rate at 84 hrs. Antibiotics delayed these time points to 72 and 120 hrs, respectively. Lung injury occurred before mortality in both groups. Polymerase chain reaction detected bacteria in the blood and lungs of all CLP mice by 24 hrs. Antibiotics delayed but did not prevent infection in CLP + Abx mice. Serum tumor necrosis factor-alpha and lung endotoxin were elevated to similar concentrations in both CLP and CLP + Abx mice. CONCLUSIONS: In this model of sepsis, antibiotics delay but do not prevent acute lung injury and mortality. Even in the presence of antibiotics, acute lung injury is strongly associated with bacteremia and bacteria within the lungs.     

Inflammatory status in sepsis alters efficacy of interleukin-18 binding protein therapy

OBJECTIVE: Sepsis remains a serious clinical problem, and multiple attempts at blocking inflammation have failed to decrease mortality rate. Interleukin-18 has been demonstrated to be an important component of the innate immune response to bacterial infections. DESIGN: Previous work demonstrated that elevated plasma concentrations of interleukin-6 obtained in the first 6 hrs of sepsis predict a worse outcome. Mice were subjected to cecal ligation and puncture and, on the basis of the plasma concentration of interleukin-6, were randomized to receive either interleukin-18 binding protein or vehicle approximately 8 hrs after the onset of sepsis. SETTING: University research laboratory. SUBJECTS: Adult, female BALB/c mice. INTERVENTIONS: We sought to determine the role of interleukin-18 in sepsis by blocking its biological activity with the interleukin-18 binding protein in the murine model of sepsis induced by cecal ligation and puncture. MEASUREMENTS AND MAIN RESULTS: In this study, elevated plasma concentrations of interleukin-6 were associated with a worse outcome. Treatment with interleukin-18 binding protein decreased inflammation as determined by lower concentrations of plasma interleukin-6 obtained 48 hrs after the onset of sepsis. In mice with increased risk of dying, interleukin-18 binding protein slightly decreased mortality rate. However, in those mice with a predicted low mortality rate, interleukin-18 binding protein significantly increased mortality rate. CONCLUSIONS: In this study, mice at low risk of death due to sepsis had decreased survival when treated with interleukin-18 binding protein. These results have potential implications for the use of interleukin-18 binding protein for treatment of chronic inflammatory conditions since it may place the host at increased risk of infectious complications.     

Immune depression in polymicrobial sepsis: the role of necrotic (injured) tissue and endotoxin

OBJECTIVE: Recent studies suggest immune dysfunction seen after the onset of polymicrobial sepsis, as produced by cecal ligation and puncture (CLP), is not caused by endotoxin (ETX) alone, but may be caused by the combined effect of the necrotic tissue (cecal ligation, [CL]) and other microbial components. Thus, the objective of this study was to assess the ability of necrotic tissue, in the presence or absence of low-dose endotoxin, to induce changes in the capacity of immune cells to produce proinflammatory or anti-inflammatory cytokines approximating those seen in CLP. DESIGN: Experimental, prospective study. SETTING: A hospital laboratory in the Center for Surgical Research. SUBJECTS: Male C3H/HeN mice. INTERVENTIONS: Mice were subjected to a CL and saline infusion (CL/Sal), CL in combination with low-dose ETX infusion (CL/ETX) (0.025 mg ETX/25 g body weight/24 hrs by a peritoneally implanted osmotic mini-pump), ETX infusion alone, saline infusion alone (Sal), CLP, or sham-CLP (Sham). Splenocytes, splenic macrophage and peritoneal macrophage were harvested from these animals 24 hrs (late) after being subjected to the above protocols. Splenocyte and macrophage inducible cytokine release was assessed by ELISA/bioassay. Survival over a 7-day period was also examined in additional groups. MEASUREMENTS AND MAIN RESULTS: Our results indicate a marked decrease in splenic interleukin (IL)-2. In addition, peritoneal or splenic macrophage IL-6 productive capacity was depressed in cells from animals subjected to CL/ETX or CLP. Alternatively, CL, in the presence or absence of ETX, induced a marked change in macrophage cytokine release capacity comparable to that seen in CLP, ie, decreased IL-12 release and increased IL-10 secretion. To the extent these cellular alterations contribute to an increase in mortality rate, we observed in subsequent survival studies that neither CL alone nor ETX produced mortality. However, the combination of CL/ETX markedly increased 7-day mortality rate (approximately 33%), although not to the same extent as CLP (80%). CONCLUSIONS: These results collectively suggest that the response to devitalized tissue produced by cecal ligation may predispose the host to the induction of a suppressive macrophage phenotype. The subsequent exposure of these animals to microbial agents induces immune dysfunction, as well as mortality seen after such a polymicrobial septic challenge.     

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.     

Caffeic acid phenethyl ester reduces mortality and sepsis-induced lung injury in rats

OBJECTIVE: Sepsis and ensuing multiorgan failure continue to be the major causes of mortality in intensive care units. Nuclear factor (NF)-kappaB activation is supposed to be one of the targets in the treatment of sepsis. We studied the effectiveness of caffeic phenethyl ester (CAPE), a known NF-kappaB inhibitor, in cecal ligation and puncture (CLP)-induced sepsis and lung injury. DESIGN: Randomized, controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Female Sprague-Dawley rats. INTERVENTIONS: CLP was performed in all rats except the rats in control and sham+CAPE groups. CAPE was administered to rats at the time of operation in sham+CAPE and CAPE+sepsis 0 groups. CAPE was administered to rats in the CAPE+sepsis12 group 12 hrs after CLP. Eight rats from each group were killed 24 hrs after CLP. Blood was taken for assessment of interleukin-1, interleukin-6, interleukin-10, and tumor necrosis factor-alpha; the right lung was removed for histopathologic examination and the left lung for biochemical examination. Apoptosis, inducible nitric oxide synthase, heat shock protein 70, malondialdehyde, catalase, superoxide dismutase, and glutathione peroxidase were studied. The rest of the rats were observed for mortality. MEASUREMENTS AND MAIN RESULTS: Mortality was significantly decreased in groups that received CAPE compared with the sepsis group. All cytokine levels were similar to control levels only in the CAPE+sepsis12 group. Apoptosis, inducible nitric oxide synthase, and heat shock protein 70 evaluation were significantly changed between all groups in the following order: control < sham+CAPE< CAPE+sepsis12 < CAPE+sepsis 0 < sepsis. Malondialdehyde and catalase were increased in the sepsis group. CONCLUSIONS: CAPE reduced mortality in sepsis and improved histopathologic variables best when it was administered after the onset of sepsis.     

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.     

Selective alpha7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis

OBJECTIVE: Tumor necrosis factor and high mobility group box 1 are critical cytokine mediators of inflammation. The efferent vagus nerve inhibits cytokine release through alpha7-nicotinic acetylcholine receptor-mediated cholinergic signaling. Here we studied whether GTS-21, a selective alpha7-nicotinic acetylcholine receptor agonist, inhibits proinflammatory cytokines in vitro and in vivo and improves survival in murine endotoxemia and severe sepsis. DESIGN: Randomized and controlled in vitro and in vivo study. SETTINGS: Research laboratory and animal facility rooms. SUBJECTS: RAW 264.7 cells and BALB/c mice treated with endotoxin or subjected to cecal ligation and puncture (CLP). INTERVENTIONS: RAW 264.7 cells were exposed to endotoxin (4 ng/mL or 10 ng/mL) in the presence or absence of GTS-21 (1-100 muM), and tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation were analyzed. Mice were treated with GTS-21 (0.4 mg/kg or 4 mg/kg, intraperitoneally) or saline 30 mins before endotoxin (6 mg/kg, intraperitoneally), and serum tumor necrosis factor was analyzed 1.5 hrs after the onset of endotoxemia. In survival experiments, mice were treated with GTS-21 (0.4 or 4.0 mg/kg, intraperitoneally) or saline 30 mins before and 6 hrs after endotoxin and then twice daily for 3 days. Severe sepsis was induced by CLP. Mice were treated with GTS-21 (4 mg/kg) or saline immediately and 6 hrs and 24 hrs after CLP, and serum high mobility group box 1 was analyzed 30 hrs after CLP. In survival experiments, GTS-21 (0.4 or 4 mg/kg) treatment was initiated 24 hrs after CLP and continued twice daily for 3 days. MEASUREMENTS AND MAIN RESULTS: GTS-21 dose-dependently inhibited tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation in vitro. GTS-21 (4 mg/kg) significantly inhibited serum tumor necrosis factor during endotoxemia and improved survival (p < .0001). GTS-21 (4 mg/kg) significantly inhibited serum high mobility group box 1 levels in CLP mice and improved survival (p < .0006). CONCLUSION: These findings are of interest for the development of alpha7-nicotinic acetylcholine receptor agonists as a new class of anti-inflammatory therapeutics.     

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.     

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.     

Carbamoyl phosphate synthase-1: a marker of mitochondrial damage and depletion in the liver during sepsis

OBJECTIVE: Mitochondrial damage and dysfunction are thought to play an important role in the pathogenesis of sepsis-induced organ failures. Unfortunately, specific markers of mitochondrial damage in vital organs do not currently exist. Recently, carbomyl phosphate synthase (CPS)-1, a protein primarily localized to liver mitochondria, was found to be present in high concentrations in the plasma of septic humans. Thus, we hypothesized that the circulatory release of CPS-1 would correlate with mitochondrial damage or impaired mitochondrial function in the liver in a clinically relevant model of sepsis. DESIGN: Prospective, randomized, controlled animal study. SETTING: University medical center research laboratory. SUBJECTS: Male, Balb/C mice, aged 10-12 wks. INTERVENTIONS: Animals were assigned to receive cecal ligation and puncture (CLP sepsis) or sham operation and compared with untreated controls. Plasma CPS-1 levels and liver mitochondrial variables, including morphology, respiratory activity, mass (i.e., cardiolipin content), and protein carbonylation, were assessed at various time points (8, 24, and 48 hrs and 6 days) after surgery. MEASUREMENTS AND MAIN RESULTS: Oxidant stress (i.e., carbonylation) was detected within 8 hrs of CLP and persisted through 48 hrs. Plasma CPS-1 levels increased dramatically at 24 hrs, remained significantly elevated at 48 hrs, and normalized by 6 days in the sepsis group. Abnormalities of liver mitochondrial morphology and function coincided with increased plasma CPS-1 levels. Mitochondrial depletion in the liver was not due to cell death but was associated with increased lysosomal clearance. Increased expression of mitochondrial biogenesis factors preceded restoration of mitochondrial variables and normalization of CPS-1 levels by day 6. CONCLUSIONS: Circulating CPS-1 is a marker of mitochondrial damage and depletion in the liver during the subacute phase of CLP sepsis. From a mechanistic standpoint, mitochondrial depletion is not due to cell death but is apparently related to the removal of damaged mitochondria by lysosomes (i.e., autophagy), followed by repletion of mitochondrial populations. Further studies are needed to determine the clinical utility of CPS-1 as a marker of sepsis severity.     

Genetic contribution to the septic response in a mouse model

The response to injury is dependent on several factors, including the type and extent of the injury, genetics, and the environment. In the present study, the genetic contribution to sepsis was evaluated in a mouse model. Sepsis was induced in two inbred mouse strains, C57BL/6J (B6) and A/J, by cecal ligation and single puncture (CLP). Frequency of mortality was significantly higher in B6 than A/J mice from 36 to 132 h after CLP. Plasma TNF-alpha, IL-1beta, and IL-6 levels were similar in both strains after CLP. IL-10 plasma levels were significantly higher in B6 mice as opposed to A/J mice after 24 h of CLP. Similarly, hepatic myeloperoxidase activity, an index of polymorphonuclear leukocytes, was elevated in B6 mice as compared with A/J mice after 24 h of CLP. On the contrary, metallothionein mRNA levels were higher in A/J mice compared with B6 mice. Finally, leptin levels were also higher in A/J than B6 mice within 19 h of CLP. This study demonstrates a genetic contribution in the response to sepsis.     

脓毒症小鼠CD4~+ CD25~+ Foxp3~+T细胞变化及其意义

目的观察脓毒症小鼠血中CD4+CD25+Foxp3+T细胞的变化特点,探讨它与脓毒症病程发展间的关系。方法选择清洁级KM雄性小鼠,用盲肠结扎穿孔术(CLP)制作脓毒症小鼠模型(CLP组),分别于2h、6h、12h、24h、48h、72h采血处死12只,并留取远端回肠HE染色光镜检查;流式细胞仪检测血中CD4+CD25+Foxp3+T细胞计数及其占CD4+T细胞的百分比,ELISA法测定细胞因子TNF-α、IL-6、IL-10的表达水平,全自动生化分析仪检测AST、ALT、BUN、Cr等肝肾功能生化指标。以假手术组(Control组)作为对照,标本采集时间点、检测方法同CLP组。结果 CLP组血CD4+CD25+Foxp3+T细胞占CD4+T细胞百分比持续增高,12h后即高于假手术组,随病程进展增高更为显著(P<0.01)。CLP2h后TNF-α水平升高即超过假手术组(P<0.01),12h到达顶峰,随后开始下降,但至CLP后72h仍高于假手术组;IL-6水平于CLP后6h出现快速升高,12h达到峰值,与假手术组相比增幅有统计学差异(P<0.01),随后下降,72h与假手术组比较无统计学差异(P>0.05);CLP2h后IL-10水平即已高于假手术组(P<0.01),12h出现快速上升改变,并持续攀升,至72h未见下降拐点。CLP后ALT、AST升高早于BUN、Cr,前者术后24h明显升高,增幅大于假手术组(P<0.01),而后者术后48h明显升高,增幅大于假手术组(P<0.01),随着时间的延长,ALT、AST、BUN、Cr均进一步升高,假手术组指标则回落到基线水平。结论 CLP小鼠模型CD4+CD25+Foxp3+T细胞比例增加与脓毒症严重程度成正比,这种变化可能是机体逃避过度的炎症反应引发严重的组织器官损伤的自身调节反应,但有一定限度,调控Treg或将为脓毒症免疫调节治疗提供新的思路。     

Glycine attenuates hepatocellular depression during early sepsis and reduces sepsis-induced mortality

OBJECTIVES: To determine whether administration of glycine, a nonessential amino acid, early after the onset of polymicrobial sepsis has any beneficial effects on hepatocellular function and the survivability of septic animals and, if so, whether the beneficial effects of glycine are associated with down-regulation of proinflammatory cytokine tumor necrosis factor-alpha production. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A university research laboratory. SUBJECTS: Male adult rats were subjected to polymicrobial sepsis by cecal ligation and puncture or sham operation followed by the administration of normal saline solution. MEASUREMENTS AND MAIN RESULTS: At 1 hr after cecal ligation and puncture, glycine (0.6 mmol/kg) or vehicle (normal saline solution) was administered intravenously over 15 mins. At 5 hrs after cecal ligation and puncture (i.e., early stage of sepsis), hepatocellular function (i.e., the maximal velocity and efficiency of in vivo indocyanine green clearance) was determined and hepatocyte injury was assessed by measuring plasma concentrations of alpha-gluthathione S-transferase. Serum tumor necrosis factor-alpha was measured by enzyme-linked immunosorbent assay. In additional animals, the necrotic cecum was excised at 20 hrs after cecal ligation and puncture, the peritoneal cavity was irrigated with saline, and the midline incision was closed in layers. Mortality was monitored for 10 days thereafter. The results indicate that hepatocellular function was depressed in the early stage of sepsis (i.e., 5 hrs after cecal ligation and puncture) as indicated by significant decreases in both maximal velocity and transport efficiency of in vivo indocyanine green clearance. Plasma concentrations of alpha-gluthathione S-transferase and tumor necrosis factor-alpha were elevated significantly at that interval after cecal ligation and puncture. Administration of glycine 1 hr after cecal ligation and puncture, however, increased maximal velocity and maximal efficiency by 60% and 101% (p <.05), respectively. Glycine administration in septic animals decreased alpha-gluthathione S-transferase and tumor necrosis factor-alpha by 43% and 80% (p <.05). In addition, glycine treatment decreased the mortality rate from 50% to 0% (p <.05) at 10 days after cecal ligation and puncture and cecal excision. CONCLUSIONS: It appears that the beneficial effect of glycine on hepatocyte function and integrity in sepsis may be mediated via down-regulation of tumor necrosis factor-alpha. Because administration of glycine attenuated hepatocellular depression and injury during early sepsis and decreased sepsis-induced mortality rates, this amino acid appears to be a useful adjunct for maintaining cellular functions and preventing lethality from polymicrobial sepsis.     

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.     

Effects of age on mortality and antibiotic efficacy in cecal ligation and puncture

The incidence and mortality of sepsis increase with age, consequently, 80% of the clinical mortality from sepsis occurs in patients over age 65. Despite this aged clinical population, most research models of sepsis use 6- to 16-week-old mice as patient surrogates. This age range of mice corresponds to human ages 10 to 17 years. To assess the influence of age on rodent CLP and on antibiotic therapy, we studied young (4 month), mature (12 month), and aged (24 month) mice. Male C57BL/6 mice (n = 27-30 in each age group) were subjected to cecal ligation and puncture (CLP), two punctures with a 25-gauge needle. Mice were observed untreated for 10 days. Young mice had 20% mortality, mature mice had 70% mortality (P = 0.0013 vs. young), and aged mice had 75% mortality (P = 0.0001 vs. young). To assess the effects of age on antibiotic therapy, mice were subjected to CLP as above (n = 38-40 in each age group). Mice were then randomized to treatment with intraperitoneal injections of ceftriaxone and metronidazole or normal saline. Therapy was initiated 12 h after CLP, and injections were repeated every 12 h for 7 days. Young mice saw a 56% decrease in mortality from CLP with antibiotic therapy (P = 0.001), and mature mice had a 30% decrease in mortality (P = 0.06). Aged mice saw no benefit from antibiotic therapy. We also compared plasma cytokine levels between young and aged mice after CLP. When compared with young mice, aged mice had higher levels of IL-6 and TNF-alpha 24 h after CLP. However, high IL-6 was predictive of mortality at any age. Mice appear to have age-dependent responses to intra-abdominal sepsis and to appropriate therapy.