Model Of Intra-abdominal Sepsis Research Articles

Host responses to sepsis vary in different low-lethality murine models.

IntroductionAnimal models for the study of sepsis are being increasingly scrutinized, despite their essential role for early translational research. In particular, recent studies have suggested that at the level of the leukocyte transcriptome, murine models of burns, trauma and endotoxemia markedly differ from their human equivalents, and are only weakly similar amongst themselves. We compared the plasma cytokine and leukocyte transcriptome responses between two different low-lethality murine models of polymicrobial intra-abdominal sepsis.MethodsSix to ten week male C57BL/6j mice underwent either the ‘gold standard’ cecal ligation and puncture (CLP) model of intra-abdominal sepsis or administration of a cecal slurry (CS), where cecal contents are injected intraperitoneally. Surviving mice were euthanized at two hours, one or three days after sepsis.ResultsThe murine leukocyte transcriptomic response to the CLP and CS models of sepsis was surprisingly dissimilar at two hours, one, and three days after sepsis. The Pearson correlation coefficient for the maximum change in expression for the entire leukocyte transcriptome that changed significantly over time (n = 19,071) was R = 0.54 (R2 = 0.297). The CS model resulted in greater magnitude of early inflammatory gene expression changes in response to sepsis with associated increased production of inflammatory chemokines and cytokines. Two hours after sepsis, CLP had more significant expression of genes associated with IL-10 signaling pathways, whereas CS had greater expression of genes related to CD28, apoptosis, IL-1 and T-cell receptor signaling. By three days, the changes in gene expression in both sepsis models were returning to baseline in surviving animals.ConclusionThese analyses reveal that the murine blood leukocyte response to sepsis is highly dependent on which model of intra-abdominal sepsis is employed, despite their similar lethality. It may be difficult to extrapolate findings from one murine model to another, let alone to human sepsis.

Protective Immunity and Defects in the Neonatal and Elderly Immune Response to Sepsis

Populations encompassing extremes of age, including neonates and elderly, have greater mortality from sepsis. We propose that the increased mortality observed in the neonatal and elderly populations after sepsis is due to fundamental differences in host-protective immunity and is manifested at the level of the leukocyte transcriptome. Neonatal (5-7 d), young adult (6-12 wk), or elderly (20-24 mo) mice underwent a cecal slurry model of intra-abdominal sepsis. Both neonatal and elderly mice exhibited significantly greater mortality to sepsis (p < 0.05). Neonates in particular exhibited significant attenuation of their inflammatory response (p < 0.05), as well as reductions in cell recruitment and reactive oxygen species production (both p < 0.05), all of which could be confirmed at the level of the leukocyte transcriptome. In contrast, elderly mice were also more susceptible to abdominal peritonitis, but this was associated with no significant differences in the magnitude of the inflammatory response, reduced bacterial killing (p < 0.05), reduced early myeloid cell activation (p < 0.05), and a persistent inflammatory response that failed to resolve. Interestingly, elderly mice expressed a persistent inflammatory and immunosuppressive response at the level of the leukocyte transcriptome, with failure to return to baseline by 3 d. This study reveals that neonatal and elderly mice have profoundly different responses to sepsis that are manifested at the level of their circulating leukocyte transcriptome, although the net result of increased mortality is similar. Considering these differences are fundamental aspects of the genomic response to sepsis, interventional therapies will require individualization based on the age of the population.

The Effects of Dexmedetomidine on Secondary Acute Lung and Kidney Injuries in the Rat Model of Intra‐Abdominal Sepsis

In the present study, the effects of dexmedetomidine on secondary lung and kidney injuries were studied in the rat model of intra-abdominal sepsis by immunohistological and biochemical examinations. We measured serum creatinine, kidney tissue malondialdehide and plasma neutrophil gelatinase-associated lipocalin levels. In order to evaluate tissue injury we determined kidney tissue mononuclear cell infiltration score, alveolar macrophage count, histological kidney and lung injury scores and kidney and lung tissue immunoreactivity scores. We demonstrated that dexmedetomidine attenuates sepsis-induced lung and kidney injuries and apoptosis in the rat model of sepsis. There is still need for comparative studies in order to determine the effects of dexmedetomidine on organ functions in early human sepsis.

P2Y12 Null Mice Are Protected Against Sepsis-Induced Lung Injury

AbstractAbstract 615The P2Y12 receptor is expressed on the platelet surface and mediates ADP-induced aggregation, but it has recently been shown to be expressed also in other cells of the immune system. Furthermore, the P2Y12 receptor gene variants correlate with pulmonary inflammation and asthma, and P2Y12 receptor deficiency or platelet depletion abrogated dust mite-induced airway inflammation, suggesting an important role for the P2Y12 receptor in pulmonary inflammation. Excessive neutrophil infiltration into the lungs is a hallmark of acute lung injury. The mechanisms that trigger this infiltration are poorly understood but may be the result of the development of a pro-inflammatory phenotype in the lung. Platelets are increasingly recognized for their ability to modulate immune responses through their interaction with neutrophils, dendritic cells, and T lymphocytes. These studies suggest an important regulatory role for the P2Y12 receptor and activated platelets in regulating neutrophil influx into the lung.Using a mouse model of intra-abdominal sepsis and acute lung injury (cecal ligation and double puncture (CLP)), we examined the role of the P2Y12 receptor in neutrophil migration and lung inflammation using P2Y12 null mice. In this study wild type (WT) and P2Y12 null (KO) mice underwent Sham surgery or CLP surgery (5 animals/group). At 24 hours post surgery, blood and lung tissue samples were collected.Our results show that the sepsis-induced increase in circulating white blood cells (WBC) was attenuated in CLP KO mice, compared with CLP WT mice (circulating WBC in CLP WT: 1.62 ± 0.41×103cell/ml* compared with CLP KO: 0.8 ± 0.4×103cell/ml, *p<0.05 WT CLP versus KO CLP mice). There were no significant differences in the WBC count in WT Sham surgery animals as compared to the KO sham surgery animals (Sham WT: 0.8 ± 0.2×103cell/ml vs. Sham KO: 0.8 ± 0.1×103cell/ml). Interestingly, no significant changes in the platelet count between the groups were observed. Next we investigated the role of P2Y12 in regulating sepsis-induced elevations in plasma levels of cytokines (TNF-a, IL-10, IL-6 and MIP-1b) (Figure 1). The concentration of each cytokine was elevated during sepsis in both WT and KO animals as compared to sham surgery animals, but, the sepsis-induced increase was significantly lower in P2Y12 null mice (**p < 0.01 KO CLP model versus WT- CLP). [Display omitted] Finally we studied neutrophil infiltration in lung tissues using hematoxylin and eosin (H & E) staining and myeloperoxidase (MPO) activity in lung tissue homogenates. Analysis of H & E staining demonstrated that the increase in inflammatory cell infiltration detected in WT was diminished in KO mice, suggesting a decrease in inflammation in the lungs of P2Y12 null mice. Furthermore, the MPO concentration was significantly decreased in KO CLP lung tissue as compared to WT CLP (Figure 2), (*p < 0.05; KO CLP model versus wild type). [Display omitted] In conclusion, our data shows a decrease in plasma cytokine levels and in circulating WBC, as well as a diminished neutrophil infiltration in the lungs indicating that P2Y12 null mice are protected against sepsis-induced lung injury. This suggests a regulatory role for P2Y12 receptor during inflammation. Disclosures:No relevant conflicts of interest to declare.

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response (1). Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47(phox), p67(phox), and p40(phox)) and Rac to a membrane-bound flavocytochrome (composed of a gp91(phox) and p22(phox) heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91(phox) -deficient mice and p47(phox)-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense (2, 13). In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase. Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion (14). Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species (15, 16). Kielland et al. (16) showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47(phox)-deficient mice. We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47(phox-/-) mice (2) in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47(phox-/-) mice enables us to delineate the specific contribution of ROS generated by p47(phox)-containing NADPH oxidase to the bioluminescent signal in these models. Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47(phox-/-) mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

Effect of therapeutic hypothermia according to severity of sepsis in a septic rat model

Aim of studyThe effects of therapeutic hypothermia (HT) during experimental sepsis may be influenced by disease severity. We experimentally investigated the effect of therapeutic HT on varying disease severity in a septic rat model. Materials and methodsAn adult male Sprague-Dawley rat model of intra-abdominal sepsis was used. To modify the disease severity, we used two different models; a moderate severe sepsis model (MSSM) and a severe septic shock model (SSSM). All rats were randomized to a hypothermia group (HT, 30–32°C) or a normothermia group (NT, 36–38°C) 1h after sepsis induction in each model. HT was maintained for 4h and rewarming was conducted for 2h. Survival time was recorded for up to 12h in the SSSM group and 24h in the MSSM group. Acute lung and liver injury, cytokine, and malondialdehyde (MDA) levels were investigated 7h after sepsis induction. Hemodynamic profiles were also evaluated. ResultsIn the SSSM, there were survival benefits and reduced acute lung and liver injury with therapeutic HT. Therapeutic HT was also associated with significantly reduced levels of plasma interleukin-6 and tissue malondialdehyde (MDA) levels in the liver and lung compared with the NT group in the SSSM. There was a tendency for the mean arterial pressure to be higher in the HT group compared to the NT group in the SSSM. In MSSM, however, there was no such beneficial effect. ConclusionIn this rat model of severe septic shock, therapeutic HT showed beneficial effects. In contrast, therapeutic HT did not show protective effect in the moderate sepsis model.

The combined effects of glutamine and growth hormone on intestinal anastomosis in the rat intra-abdominal sepsis model

AimIntestinal anastomoses are always risky in patients who develop intra-abdominal sepsis. In this study, the effects of combined glutamine and growth hormone (GH) on healing of intestinal anastomosis following intestinal repair in the rat intra-abdominal sepsis was induced. Material and methodsForty Sprague Dawley Albino rats at 10 weeks weighing between 180 and 240 g were included in the study. All the animals were divided into five groups comprising eight rats each. In the control group, no treatment was given in addition to the routine oral nutrition before and after surgery. In the other groups, following surgery, oral glutamine was given at a dose of 1 mg/kg/d in the glutamine group, subcutaneous GH was given at a dose of 1 mg/kg/d in the GH group, and combined glutamine and GH were administered at the same doses in the glutamine + GH group. In rats, a clinical model mimicking intestinal fistula was generated and fistula repair was performed, and the bursting pressure of the repair area and tissue hydroxyproline level of the repair area were calculated. ResultsCompared with the control group, glutamine, GH, and combined groups displayed significantly higher mean bursting pressures and tissue hydroxyproline levels. ConclusionIn order to decrease the risks originating from impaired mechanisms due to intra-abdominal sepsis, and to make anastomosis safer, combined use of glutamine and GH increases the bursting pressure of anastomosis. While the use of either of these two substances alone is effective, combined use makes this effect more prominent.

Cecal Ligation and Puncture Followed by Methicillin-Resistant Staphylococcus aureus Pneumonia Increases Mortality in Mice and Blunts Production of Local and Systemic Cytokines

Mortality in the intensive care unit frequently results from the synergistic effect of two temporally distinct infections. This study examined the pathophysiology of a new model of intra-abdominal sepsis followed by methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Mice underwent cecal ligation and puncture (CLP) or sham laparotomy followed 3 days later by an intratracheal injection of MRSA or saline. Both CLP/saline and sham/MRSA mice had 100% survival, whereas animals with CLP followed by MRSA pneumonia had 67% 7-day survival. Animals subjected to CLP/MRSA had increased bronchoalveolar lavage concentrations of MRSA compared with sham/MRSA animals. Animals subjected to sham/MRSA pneumonia had increased bronchoalveolar lavage levels of interleukin 6 (IL-6), tumor necrosis factor α, and granulocyte colony-stimulating factor compared with those given intratracheal saline, whereas CLP/MRSA mice had a blunted local inflammatory response with markedly decreased cytokine levels. Similarly, animals subjected to CLP/saline had increased peritoneal lavage levels of IL-6 and IL-1β compared with those subjected to sham laparotomy, whereas this response was blunted in CLP/MRSA mice. Systemic cytokines were upregulated in both CLP/saline and sham/MRSA mice, and this was blunted by the combination of CLP/MRSA. In contrast, no synergistic effect on pneumonia severity, white blood cell count, or lymphocyte apoptosis was identified in CLP/MRSA mice compared with animals with either insult in isolation. These results indicate that a clinically relevant model of CLP followed by MRSA pneumonia causes higher mortality than could have been predicted from studying either infection in isolation, and this was associated with a blunted local (pulmonary and peritoneal) and systemic inflammatory response and decreased ability to clear infection.

Peritoneal fluid as a novel mechanism for neutrophil priming in a clinically relevant model of intra-abdominal sepsis

Peritoneal fluid as a novel mechanism for neutrophil priming in a clinically relevant model of intra-abdominal sepsis

Evaluation of the Effects of Laparotomy and Laparoscopy on the Immune System in Intra-Abdominal Sepsis—A Review

This review portrays the most common experimental models of intra-abdominal sepsis. Additionally, it outlines the facts that distinguish laparotomy from laparoscopy, in respect to the immune response, when comparing these two techniques in experimental models of intra-abdominal sepsis. It describes the consequences of pneumoperitoneum and trauma produced by laparoscopy or laparotomy, respectively, on bacterial translocation and immunity. Furthermore, we report the few efforts that have been made in clinical settings, where surgeons have attempted to utilize laparoscopy as a therapeutic module when treating peritonitis or sepsis of abdominal origin. Certainly there is a need for more research in order to fortify the role of pneumoperitoneum in sepsis of abdominal origin. It seems that minimally invasive surgery will inevitably gain acceptance by surgeons, as evidence points that by inflicting less trauma the healing response is expected to be more efficient, especially in septic patients.

Effects of the Antimicrobial Peptide LL-37 and Hyperthermic Preconditioning in Septic Rats

The authors tested the effects of LL-37 prophylaxis or therapy on the outcome after intraabdominal sepsis and examined whether hyperthermic preconditioning plus LL-37 therapy augments host immune response and improves survival. A rat model of peritoneal contamination and infection (PCI) with human stool was used to simulate clinical conditions. In trial 1, the authors compared (1) PCI, (2) LL-37 prophylaxis (0.5 mg/kg, 12 h before PCI), and (3) LL-37 therapy (0.5 mg/kg, 1 h after PCI). In trial 2, the authors compared (1) PCI, (2) LL-37 therapy, (3) hyperthermic preconditioning (41 degrees C for 1 h, 24 h before PCI), and (4) LL-37 therapy and hyperthermic preconditioning. The primary endpoint was mortality at 120 h. In trial 2, secondary endpoints were systemic levels of tumor necrosis factor alpha, interleukin 6, macrophage inflammatory protein 2, and heat shock protein 70; leukocyte counts; and neutrophil granulocyte phagocytosis. In trial 1, 30% of the control group compared with 70% of the LL-37 therapy group survived, but 55% after LL-37 prophylaxis survived (P = 0.038). In trial 2, 38% of the controls, 67% of the LL-37 therapy, 59% of the hyperthermic preconditioned, and 90% of the hyperthermic preconditioned plus LL-37 therapy group survived (P = 0.01). LL-37 therapy plus hyperthermic preconditioning reduced proinflammatory cytokine concentrations after sepsis; specifically compared with controls, macrophage inflammatory protein-2 and interleukin-6 levels were 1.5 +/- 1.5 versus 11 +/- 6 pg/ml (P = 0.028) and 13 +/- 8 versus 86 +/- 31 pg/ml, (P = 0.015), respectively. In this model of intraabdominal sepsis, LL-37 therapy improved outcome. Hyperthermic preconditioning per se was not successful, but in combination with LL-37 therapy, the survival rate after sepsis was increased and the proinflammatory cytokine response was downgraded.

Combination of Fibrin Glue with Growth Hormone Augments Healing of Incomplete Intestinal Anastomoses in a Rat Model of Intra-Abdominal Sepsis: A Dynamic Study

This study was devised to investigate whether fibrin glue (FG) in combination with growth hormone (GH) could have a beneficial effect at a late period (14 days) after injury. Male Wistar rats, with abdominal sepsis induced by an incomplete anastomosis, were divided into three groups. In the control group, the rats got incomplete anastomoses sutured alone; in the FG and FG/GH groups, anastomoses protection was performed with application of FG alone or in combination with GH. The anastomotic bursting pressure (ABP) was significantly higher in the FG/GH group than that of the FG group on postoperative day (POD) 5 (p <. 01), while it could not be measured from POD 7 to POD 14 because of intestinal dehiscence. There was no difference between FG and FG/GH group on POD 3 and POD 5 in anastomotic tensile strength, which was significantly higher in the FG/GH group than that of the FG group from POD 7 to POD 14 (p <. 001). Hydroxyproline content of the FG/GH group was significantly higher than that of the control from POD 3 and that of the FG group from POD 5 (p <. 05). Combination of FG with GH had a synergistic effect to improve intestinal anastomotic healing over a limited 14-day course of observation.

Effect of the Combination of Fibrin Glue and Growth Hormone on Incomplete Intestinal Anastomoses in a Rat Model of Intra-Abdominal Sepsis

The presence of established intra-abdominal sepsis has been considered a contraindication to primary anastomoses. Our hypothesis was that fibrin glue (FG), growth hormone (rhGH), and combination of them synergistically improve intestinal primary anastomotic healing in a rat model of intestinal fistulae with peritonitis. Male Wistar rats, induced intestinal fistulae with peritonitis after 24 h, were performed an enterectomy and intestinal anastomoses. Group A, rats (n = 60) had a complete anastomoses (end-to-end single layer anastomoses using 12 inverted interrupted 6-0 sutures) without peritonitis, group B, rats (n = 60) had a complete anastomoses after 24 h of peritonitis, group C rats had an incomplete anastomoses (four inverted interrupted sutures), groups D, E, F rats (n = 60) received FG, rhGH, or both of them, respectively. rhGH was given daily for 5 days. Anastomoses indicated the anastomotic bursting pressure (ABP), tensile strength, and hydroxyproline content, were determined. On POD 1, ABP of group C and group D was significantly lower than that of other groups (P < 0.01); On POD 3, ABP could not be determined because of intestinal dehiscence in groups C and E, ABP was significantly higher in groups D and F than that of groups A and B (P < 0.01); the ABP increased after 5 days of operation in groups A, B, and F. At the same time, that of group D decreased (P < 0.01). On POD 5, the tensile strength was significantly higher in groups A, D, and F than that in groups C, and E. On POD 5, hydroxyproline content was higher in groups D and F compared to that in group C (P < 0.05). These data suggested that FG improve intestinal primary anastomotic healing within post-operative 5 days in a rat model of intestinal fistulae with peritonitis. RhGH alone fails to improve intestinal anastomotic healing, and the combination of FG and rhGH have no synergistic effect to improves intestinal anastomotic healing.

Efficacy of moxifloxacin monotherapy versusgatifloxacin monotherapy, piperacillin-tazobactam combination therapy, and lindamycin plus gentamicin combination therapy: An experimental study in a rat model of intra-abdominal sepsis induced by fluoroquinolone-resistant Bacteroides fragilis

Background: In intra-abdominal infections, the activity of antimicrobial agents against Bacteroides fragilis and phenotypically related organisms, and the increasing resistance of these organisms, are of particular importance and concern to surgeons. In vitro data suggest that moxifloxacin is more active than other quinolones against obligately anaerobic organisms, including Bacteroides spp. Objective: The aim of this study was to compare the efficacy of moxifloxacin monotherapy versus gatifloxacin monotherapy and 2 combination therapies (piperacillin-tazobactam and clindamycin plus gentamicin) in a rat model of intra-abdominal sepsis. The end point was marked by the incidence of mortality and intra-abdominal abscesses at necropsy 7 days after bacterial challenge. Methods: Three different strains of B fragilis with different degrees of resistanceto moxifloxacin (minimum inhibitory concentrations [MICs]: 4, 8, and 16 pg/mL) were added to the challenge inoculum in 3 separate experiments. Groups of 20 animals were used in each experiment. Group 1 served as saline-treated controls; group 2 received moxifloxacin 15 mg QD; group 3 received gatifloxacin 25 mg QD; group 4 received piperacillin-tazobactam 93 mg (−83 mg of piperacillin) QD; and group 5 received a combination of clindamycin 15 mg TID plus gentamicin 2 mg TID. All treatments were given intramuscularly. For all antimicrobials, dose was based on peak and trough serum drug concentrations determined by prior testing, with animal doses adjusted based on the ratio of body surface area to body weight, and comparing these doses and levels with studies in humans. Results: In all 3 experiments, the mortality rate with moxifloxacin was significantlylower or statistically similar compared with antibiotic active comparators ( P ≤ 0.024). In addition, there were no significant differences in the incidence of abscess with moxifloxacin versus its comparators or between the 3 moxifloxacin groups across experiments. The best results for moxifloxacin were found in the experiment in which the B fragilis strain with MIC 16 μg/mL was added to the inoculum. Conclusion: The results of this study in an animal model of intra-abdominalsepsis induced by fluoroquinolone-resistant B fragilis suggest that moxifloxacin monotherapy performs as well as combination regimens such as piperacillin-tazobactam and clindamycin plus gentamicin, and is as effective as other fluoroquinolones with antianaerobic activity, such as gatifloxacin.

Effects of G-CSF and antibiotic prophylaxis in a 2 x 2 factorial design on outcome in septic rats.

In a recently completed randomised clinical trial in patients with colorectal cancer resections the combination of the granulocyte-colony stimulating factor (G-CSF) + cefuroxime/ metronidazole (cef/met) was superior to ofloxacin/metronidazole (ofl/met). These combinations were used to confirm the clinical data and to validate the concept of clinic modelling randomised trials (CMRTs) in a rat model of intra-abdominal sepsis. 80 male Wistar rats were randomised in a 2 x 2 factorial study design. All animals (n = 20/group) received anaesthesia, antihistamines, antibiotic prophylaxis, peritoneal contamination and infection. Groups were: 1) G-CSF + cef/met; 2) placebo + cef/ met; 3) G-CSF+ofl/met; 4) placebo + ofl/met. G-CSF (20 g/kg) prophylaxis was applied three times. Survival at 120 h was analysed with the Kaplan Meier method. Survival rate was best in the G-CSF + cef/met group with 75% and was significantly improved compared to the cef/met placebo group, in which only 42% survived (P < 0.05). Survival rate between both G-CSF groups was similar being 75% in the cef/met and 72% in the ofl/met group. P = 0.10). Ozone increased TNF-alpha and MIP-2 after infection: 127 +/- 23 pg/ml and 94 +/- 19 pg/ml (control group: 398 pg/ml and 369 pg/ml; P < 0.002 and P < 0.01). IL-6 levels were similar in both groups. The results of this CMRT confirmed the result of our clinical G-CSF trial in that G-CSF prophylaxis was most efficacious in combination with cef/met to improve the outcome.

The effect of hyaluronan-based agents on adhesion formation in an intraabdominal sepsis model.

Because of the technical difficulty during subsequent surgical intervention, adhesion remains the most important predictor of outcome in the staged procedures following emergent colorectal surgery. The aim of this study was to assess the long-term effects of hyaluronan (HA)-based adhesion barriers 3 months following the infectious insult to the peritoneal cavity. Wistar albino rats were divided into three sham and four infectious groups, each consisting of 20 rats. Sham groups consisted of a control group (I), which had undergone manipulation of the cecum in the first operation and saline irrigation following the cecal resection in the second operation; an HA-based bioresorbable membrane (BM) group (II), in which 20 x 20- and 35 x 25-mm pieces of HA-based bioresorbable membrane were placed over the cecectomy area and under the midline incision, respectively, with other conditions the same as for the control (I) group; and an HA-based solution (S) group (II), where HA solution was used as an irrigation solution, with other conditions the same as for the control group (I). Infectious groups consisted of a septic group (IV), which had undergone cecal ligation and puncture in the first operation and saline irrigation following the cecal resection in the second operation; a sepsis + HA-BM group (V), in which two sheets of membrane were applied, with other conditions the same as for the septic group (IV); a sepsis + HA-S group (VI), in which HA solution was used as an irrigation solution, with conditions otherwise the same as for the septic group (IV); and a sepsis + HA-BM + HA-S group (VII), where HA solution was used as an irrigation solution and two sheets of membrane were applied, with other conditions the same as for the septic group (IV). At the end of the 3-month period, mortality, septic complications, and intraabdominal adhesions (adhesion scores and adhesion tensile strength measurements) were recorded. Significantly denser adhesions were found in the septic group (IV) when compared to the others (P < 0.001). HA-S significantly reduced not only the incidence of adhesion but also the adhesion tensile strength in infectious groups (P < 0.001 compared to the sepsis group). However, HA-BM significantly reduced adhesion tensile strength only. Irrigation of the peritoneal cavity with HA-S may prove to be useful to shorten the reversal time period and decrease morbidity following staged procedures for intraabdominal sepsis.

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.

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. Prospective, randomized, controlled study. Animal laboratory in a university medical center. Male C57BL/6 mice, 6-10 wks old. 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. 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. 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.

Deleterious effects of mild hypothermia in septic rats are ameliorated by granulocyte colony-stimulating factor.

The authors studied the effects of mild hypothermia on the outcome in a rat model of intraabdominal sepsis and tested whether granulocyte colony-stimulating factor (G-CSF) augments the host response and improves outcome during mild hypothermia. A rat model of peritoneal contamination and infection with human stool bacteria was used to simulate clinical trials that included increasing complexity. In trial 1, postoperative hypothermia (32 degrees C) was compared with normothermia (38 degrees C), without supportive treatment (10 rats per group). In trial 2, with a more severe infection, rats were given antibiotic prophylaxis. Using 20 rats per group, the authors compared postoperative hypothermia (32 degrees C), normothermia, and postoperative hypothermia (32 degrees C) with 20 mug/kg G-CSF prophylaxis given 12 h before surgery and 12 h and 36 h after surgery. The primary endpoint was death at 120 h. Secondary endpoints were systemic cytokine concentrations, leukocyte counts, and the phagocytic activity of granulocytes and monocytes. In trial 1, 50% of the normothermia group and 10% of the postoperative hypothermia group survived. In trial 2, 50% of the normothermia group, 20% of the hypothermia group, and 60% of the hypothermia plus G-CSF group survived. Postoperative hypothermia plus G-CSF reduced plasma concentrations of interleukin-6 (hypothermia group, 511 +/- 104 pg/ml; hypothermia plus G-CSF group, 247 +/- 51 pg/ml) and macrophage inflammatory protein-2 (hypothermia group, 239 +/- 43 pg/ml; hypothermia plus G-CSF group, 178 +/- 21 pg/ml). In this rat model of intraabdominal sepsis, postoperative hypothermia was deleterious. However, G-CSF treatment, initiated before contamination, reduced the mortality rate, increased the neutrophil count, and downgraded the systemic cytokine response.

Effects of aging on mortality, hypothermia, and cytokine induction in mice with endotoxemia or sepsis

Aging is accompanied by an altered stress response that underlies increased susceptibility of the elderly patients to physiological stress such as infection and sepsis. In the present study, we investigated the effects of aging on mortality, hypothermia, and cytokine induction in mouse models of intra-abdominal sepsis and endotoxemia. Systemic inflammation associated with either cecal ligation/puncture (CLP) or injection with bacterial endotoxin, lipopolysaccharide (LPS), resulted in a significantly elevated mortality rate in aged (24 months) compared to young (4 months) mice. The aged mice also showed profound hypothermia during these inflammatory stresses; the severity of hypothermia at the early phase of sepsis or endotoxemia could predict the mortality of individual animals. The stress-mediated induction of interleukin-1β, interleukin-6, and interleukin-10 (IL-1β, IL-6, and IL-10) in the circulating blood tended to be higher with aging in both CLP and LPS models, and in particular, the induction of IL-6 was significantly augmented with aging. The serum level of IL-6 showed a strong correlation with degrees of hypothermia. In the heart and lungs, the induction of mRNA for IL-6 and IL-10 was also significantly enhanced with aging. These results clearly demonstrate an age-associated increase in mortality, hypothermia, and induction of IL-6 during endotoxemia and sepsis.

Iron overload before cecal ligation and puncture increases mortality.

Iron metabolism is dysregulated in critically ill patients. A mouse model of dysregulated iron metabolism was used to examine the consequence of iron loading upon sepsis. Mice deleted in the hfe gene (hfe-/-) abnormally accumulate iron in tissue; defects in the human hfe gene are clinically expressed as hemochromatosis. Hfe-/- mice and wild-type counterparts were randomized to receive either high- or low-iron diets for 2 weeks. After iron loading, mice were subjected to cecal ligation and puncture (CLP), a clinically relevant animal model of intra-abdominal sepsis. A preliminary (but underpowered) study suggested that iron-loaded hfe-/- mice had increased mortality as compared with hfe-/- mice fed a low-iron diet. There was no difference between wild-type and hfe-/- mice fed a low-iron diet or between wild-type mice fed hihg- and low-iron diets. A subsequent, appropriately powered study showed that iron-loaded hfe-/- mice had significantly higher mortality from intra-abdominal sepsis than hfe-/- mice fed a low-iron diet. Iron loading was confirmed through chemical assay of iron concentration in hepatic tissue. Animals with dysregulated iron handling, loaded with iron and subjected to CLP, had double the mortality of animals with normal iron levels. Critical care patients often have altered iron metabolism. In clinical practice, critically ill patients may receive iron through direct administration and the transfusion of blood products. Iron therapy may adversely affect the clinical outcome from sepsis.