Cecal Ligation And Puncture Procedure Research Articles

Dexamethasone effects on vascular flow and organ injury in septic mice

BackgroundTo demonstrate the effects of low-dose dexamethasone treatment on mesenteric artery blood flow, oxidative injury, vascular reactivity, and survival in Swiss albino mice with intra-abdominal polymicrobial sepsis accomplished by cecal ligation and puncture (CLP). MethodsMice were allocated to CLP + saline, CLP + dexamethasone, sham + saline, and sham + dexamethasone subgroups to evaluate blood flow, organ injury, and vascular response to consecutive phenylephrine administrations at 24, 48, and 72 h. Survival rates were also evaluated in a different group of mice. Dexamethasone (1 mg/kg/d) and saline (4 mL/kg/d) were administered intraperitoneally to mice 2 h after CLP or sham procedure, whichever appropriate, and repeated once a day until evaluation time at 48 and 72 h. Relaparotomy was performed at the concerned time and mesenteric blood flow was measured, and liver, lung, and peritoneum samples were obtained. Alteration in mesenteric blood flow response to intravenous phenylephrine injections was recorded at the related time intervals in different mice groups. Survival group was followed up by 7-d administration of dexamethasone or saline for 18 d. ResultsThe significant fall in mesenteric blood flow after CLP ameliorated with dexamethasone treatment at 48 and 72 h. Dexamethasone also diminished the malonyl dialdehyde level, which is an indicator of organ injury raised after CLP, at 24 h in liver, lung, and peritoneum samples. Dexamethasone therapy has significantly enhanced the vascular response to phenylephrine injections at all doses; however, no change was observed in survival rates. ConclusionsLow-dose dexamethasone has beneficial effects on mesenteric blood flow and organ injury in experimental sepsis models.

Examination of enzymes concentration in the blood of rats with sepsis caused by mixed and pure bacterial cultures

A clinical form of sepsis induced by cecal ligation and puncture (CLP) was caused in order to monitor the concentration of enzymes (alanine aminotransferase - ALT, aspartate aminotransferase - AST, lactate dehydrogenase - LDH, amylase and creatine kinase - CK) in the rat blood. Experiments were performed on male Wistar rats, weighing on average 215±25 g. The rats were divided into four groups. In the first three groups (n=28 per group), sepsis was induced by pure culture of Escherichia coli (EC) or Staphylococcus aureus (SA) and mixed culture (MK) of caecum, while the fourth group included 20 control rats who underwent an abdominal incision. Blood was taken in time intervals of 12, 24, 72 and 120 hours. During the experimental protocol, we identified significant changes of all monitored enzymes in the serum of infected rats. After a period of 12 hours there was a significant increase in ALT (all rats with sepsis), AST and LDH (rats in the MK group) levels, while a decrease was noted in the concentration of amylase (EC, SA). Similarly, 24 hours after the CLP procedure, a significant decrease of amylase (MK) and AST (SA) was recorded, while serum LDH level varied significantly from elevated (EC, SA) to reduced (MC) values. Finally, at the time intervals of 72 and 120 hours the concentration of nearly all monitored enzymes has shown a decline, while significance was noted in lowering of ALT (MK), AST (SA, EC) and amylase (SA) levels. Statistical significance could not be observed in the change of CK levels at any of examined time points.

Role of Peroxynitrite in Sepsis-Induced Acute Kidney Injury in an Experimental Model of Sepsis in Rats

The mechanisms involved in sepsis-induced acute kidney injury (AKI) are unknown. We investigated the role of nitrosative stress in sepsis-induced AKI by studying the effects of manganese (III) tetrakis-(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), a peroxynitrite decomposition catalyst, and aminoguanidine (AG), a selective nitric oxide synthase 2 (NOS2) inhibitor and peroxynitrite scavenger, on kidney function of rats subjected to cecal ligation and puncture (CLP). Sprague-Dawley rats (weighing 350 [SD, 50] g) were treated with MnTMPyP (6 mg/kg i.p.) or AG (50 mg/kg i.p.) at t = 12 and 24 h after CLP or sham procedure. At t = 36 h, mean arterial pressure and aortic blood flow were measured, and blood and urine samples were obtained for biochemical determinations, including creatinine clearance, fractional excretion of sodium, and neutrophil gelatinase-associated lipocalin concentration in the urine. Kidney tissue samples were obtained for (i) light microscopy, (ii) immunofluorescence and Western blot for 3-nitrotyrosine and NOS2, (iii) gene expression (quantitative real-time polymerase chain reaction) studies (NOS1, NOS2, NOS3, and superoxide dismutase 1), and (iv) matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Mean arterial pressure was unchanged and aortic blood flow decreased 25% in CLP animals. The sepsis-induced (i) decreased urine output and creatinine clearance and increased fractional excretion of sodium and urinary neutrophil gelatinase-associated lipocalin concentration, (ii) increased protein nitration and NOS2 protein, and (iii) NOS1 and NOS2 upregulation were all significantly attenuated by treatment with MnTMPyP or AG. Nitrated proteins in renal tissue from CLP animals (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) were glutamate dehydrogenase, methylmalonate-semialdehyde dehydrogenase, and aldehyde dehydrogenase, mitochondrial proteins involved in energy metabolism or antioxidant defense. Nitro-oxidative stress is involved in sepsis-induced AKI, and protein nitration seems to be one mechanism involved.

Anticancer Agent 2-Methoxyestradiol Improves Survival in Septic Mice by Reducing the Production of Cytokines and Nitric Oxide

Cytokine production is critical in sepsis. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, inhibits hypoxia-inducible factor 1α (HIF-1α) and is an antiangiogenic and antitumor agent. We investigated the effect of 2ME2 on cytokine production and survival in septic mice. Using i.p. LPS or cecal ligation and puncture (CLP), sepsis was induced in BALB/c mice that were simultaneously or later treated with 2ME2 or vehicle. Twelve hours after the LPS injection, serum and peritoneal fluid cytokine and nitric oxide (NO) levels were analyzed using enzyme-linked immunosorbent assay and the Griess reaction. Lung injuries were histologically analyzed, and liver and kidney injuries were biochemically analyzed. Survival was determined 7 days after LPS injection or CLP procedure. In vivo and in vitro effects of 2ME2 on LPS-induced macrophage inflammation were determined. The effect of 2ME2 on HIF-1α expression, nuclear factor κB (NF-κB), and inducible NO synthase (iNOS) in LPS-treated RAW264.7 cells, a murine macrophage cell line, was determined using Western blotting. 2-Methoxyestradiol treatment reduced LPS-induced lung, liver, and kidney injury. Both early and late 2ME2 treatment prolonged survival in LPS- and CLP-induced sepsis. 2-Methoxyestradiol significantly reduced IL-1β, IL-6, TNF-α, and NO levels in septic mice as well as in LPS-stimulated peritoneal macrophages. 2-Methoxyestradiol treatment also reduced the LPS-induced expression of HIF-1α, iNOS, and pNF-κB in RAW264.7 cells, as well as iNOS and pNF-κB expression in siHIF-1α-RAW264.7 cells. 2-Methoxyestradiol prolongs survival and reduces lung, liver, and kidney injury in septic mice by inhibiting iNOS/NO and cytokines through HIF-1α and NF-κB signaling.

A newly synthetic vitamin E derivative, E-Ant-S-GS, attenuates lung injury caused by cecal ligation and puncture-induced sepsis in rats

Cytokine activation and the ensuing spread of damage to distant organs play a central role in sepsis caused by generalized peritonitis, which accompanies surgical conditions such as gastrointestinal perforation. Anti-inflammatory properties have been discovered in endogenous substances such as vitamin E; we evaluated, in a rat model of peritonitis-induced sepsis, the newly synthetic vitamin Ederivative E-Ant-S-GS, in which the endogenous substances vitamin E, glutathione, 5-OH-anthranilic acid, and succinic acid are chemically linked. We used a model of sepsis in male Wistar rats with the cecal ligation and puncture (CLP) method. To evaluate the anti-inflammatory effects of E-Ant-S-GS, we measured serum interleukin-6 (IL-6) levels at various times after CLP. To assess the effects of E-Ant-S-GS in acute lung injury, we evaluated histologically lung tissue 12 hours after CLP by hematoxylin-eosin staining. In addition, myeloperoxidase (MPO) activity and expression of protease-activated receptor 1 (PAR1) and high mobility group box 1 (HMGB1) in the lung were determined. Serum IL-6 levels increased progressively after the CLP procedure; this cytokine induction was attenuated by E-Ant-S-GS. Increased MPO activity in lung tissue and marked changes in lung histology caused by CLP-induced sepsis were also ameliorated by E-Ant-S-GS. In addition, E-Ant-S-GS suppressed the upregulation of PAR1 and HMGB1 in the lungs after CLP. The newly synthetic vitamin E derivative E-Ant-S-GS showed anti-inflammatory actions and organ-protective effects in a rat model of sepsis, suggesting its potential clinical use as a therapeutic agent against systemic inflammation.

Nonhematopoietic toll-like receptor 2 contributes to neutrophil and cardiac function impairment during polymicrobial sepsis.

Toll-like receptor 2 (TLR2) has been implicated in neutrophil and cardiac dysfunction during sepsis. Here we tested the hypothesis that nonhematopoietic (parenchymal) and hematopoietic TLR2 play distinct roles in sepsis pathogenesis. To achieve this, we generated two groups of chimeric mice with TLR2 deletions either in nonhematopoietic cells (knockout [KO] mice with wild-type [WT] bone marrow [BM]) or in BM cells (WT mice with KO-BM). Polymicrobial sepsis was created by cecal ligation and puncture (CLP). Neutrophil functions, cytokine production, and bacterial clearance were investigated following CLP or sham procedures. Cardiac contractile function was measured in a Langendorff apparatus. Intracellular reactive oxygen species (ROS) were measured using redox-sensitive dye and flow cytometry. Cecal ligation and puncture mice had markedly increased peritoneal neutrophil recruitment compared with the sham-operated mice. Toll-like receptor 2 KO mice, regardless their TLR2 phenotypes (WT vs. KO) in their BM-derived hematopoietic cells, had markedly increased neutrophil migration as well as phagocytosis and reduced cytokine productions compared with TLR2 WT mice following polymicrobial peritonitis. These changes in the chimeric TLR2 KO mice were associated with enhanced blood bacterial clearance and markedly improved cardiac contractile function. Moreover, CLP induced a robust ROS production in the peritoneal leukocytes isolated from WT mice but not from TLR2 KO mice. Taken together, these data indicate that TLR2, particularly that of nonhematopoietic cells, plays a major role in sepsis pathogenesis by impairing neutrophil migratory and phagocytic function, promoting cytokine production, and mediating cardiac contractile dysfunction during polymicrobial sepsis. Toll-like receptor 2 also mediates critical ROS production during polymicrobial sepsis.

Liver X Receptor Protects against Liver Injury in Sepsis Caused by Rodent Cecal Ligation and Puncture

Liver X receptor (LXR) is a transcription factor of the nuclear receptor family, regulating genes involved in metabolism, inflammation, and apoptosis. In the present investigation, we examined the role of LXR in organ injury and systemic inflammation in rodent models of polymicrobial peritonitis caused by cecal ligation and puncture (CLP). Rats were subjected to CLP sepsis or a sham operation. Some were treated with the synthetic LXR agonist GW3965 0.3 mg/kg 30 min prior to the CLP procedure, and organs and plasma were harvested at 3, 10, 18, or 24 h. Organs were analyzed for RNA expression by quantitative polymerase chain reaction or for morphologic differences by histologic review. Organ injury and inflammatory markers were measured in plasma. Expression of the LXRα gene was decreased in the livers of CLP rats compared with sham-operated rats. Administration of a synthetic agonist of LXR (GW3965) reduced biochemical indices of liver injury in the blood of CLP rats. We also demonstrated that liver injury associated with CLP is aggravated in LXRα- and LXRαβ-deficient mice compared with wild-type and LXRβ-deficient mice, indicating a role for LXRα in protecting the liver. The enhanced liver injury in LXR-deficient mice was associated with elevated plasma concentrations of high mobility group box 1, a late mediator of inflammation and a known factor in the pathology of this model. Collectively, these results argue in favor of a role for LXRα in protection against liver injury in experimental sepsis induced by CLP.

Anesthesia Literature Review

Anesthesia Literature Review

NEUTROPHIL ELASTASE INHIBITOR IMPROVES SURVIVAL OF RATS WITH CLINICALLY RELEVANT SEPSIS

Sivelestat sodium hydrate is a selective inhibitor of neutrophil elastase, which is effective in acute lung injury associated with systemic inflammatory response syndrome. However, the effectiveness of sivelestat in sepsis has not been fully examined. In the present study, the effect of sivelestat on severe sepsis in a rat cecal ligation and puncture (CLP) model was investigated. Adult male Sprague-Dawley rats underwent CLP and were randomly divided into two groups: sivelestat-treated group and saline-treated controls. The serum concentrations of several inflammatory mediators were measured. Hematoxylin-eosin staining, and immunohistochemical staining for high-mobility group box chromosomal protein 1 (HMGB1), IL-8, and CD68 were performed on the lungs to assess pathological changes found 12 h after the CLP procedure. Treatment with sivelestat significantly improved the survival rate of the post-CLP septic animals (P = 0.030). Sivelestat also induced a significant reduction in serum IL-1beta (P = 0.038) and IL-10 (P = 0.008) levels in these CLP rats. Serum HMGB1 levels had no significant difference between the sivelestat-treated and the control group. The lungs from sivelestat-treated rats exhibited less severe pathological changes and decreased the numbers of HMGB1, IL-8, and CD68-positive cells (P < 0.001). Sivelestat significantly improved survival rate of rats with clinically relevant sepsis, possibly by attenuating sepsis-induced systemic inflammatory response and lung injury. This may explain the implicated health benefits of sivelestat in reducing morbidity and mortality from sepsis.

The impact of crystalloid and colloid infusion on the kidney in rodent sepsis

Volume replacement remains one of the pillars of sepsis therapy. The effect of different volume solutions on kidney function in sepsis still remains unclear. We therefore determined the impact of crystalloid and colloid solutions on kidney function in a rodent model of abdominal sepsis induced by cecal ligation and puncture (CLP). Anesthetized rats underwent the CLP procedure, whereas control animals were sham operated. Septic animals were treated with crystalloid and colloid solutions. Hemodynamic variables and blood gases were measured. After 24 h animals were re-anesthetized, the kidneys were harvested, and creatinine (crea), urea, cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) were investigated. Septic animals exhibited a mortality rate of 19% after 24 h. Gelatin-treated animals showed significantly increased levels of crea and urea. Colloids [gelatin 4% (Gel) or hydroxyethyl starch 6% 130/0.4 (HES)] as volume replacement resulted in elevated levels of NGAL. The histopathological observations revealed that Gel- and HES-treated animals showed vesicles within epithelial cells of the tubulus system and an overall increased injury. In contrast, total injury scores in groups treated with crystalloids [0.9% NaCl (NaCl) and Sterofundin ISO (SteroIso)] were not significantly different compared to sham-treated animals. None of the examined volume solution was inert to the kidney. In a CLP rodent sepsis model, animals infused with balanced crystalloid SteroIso exhibited the least effects on kidney function. Both hydroxyethyl starch 6% 130/0.4 and gelatin 4% derogated the kidney, whereas gelatin was more harmful when compared with hydroxyethyl starch.

Changes of myocardial apoptosis,Bcl-2 and Caspase-3 in septic rats

Objective To investigate myocardial apoptosis in the septic rats and to evaluate the role of Bcl-2, Caspase-3 in signal transduction of myocardial apoptosis. Methods Sixty Wistar rats were randomly divided into control group (n=30) and septic group (n=30). Either group was then divided into 5 subgroups:3 h,6 h,12 h,24 h and 36 h subgroup. Sepsis model was established by cecal ligation and puncture (CLP). Control group were given the same treatments except CLP. TUNEL method was used to determine apoptosis index (AI). Western blot was applied to determine Bcl-2 and Caspase-3 expression. Results (1) AI value increased progressively after CLP procedure and peaked at 36 h(43.742±2.495), which showed significant difference compared with control group(4.311±0.035 ). There was no significant difference between 24 h and 36 h subgroup (P>0.05). (2) Bcl-2 protein levels significantly decreased after CLP procedure compared with control group (P 0.05). Caspase-3 levels gradually increased after CLP procedure,and 36 h subgroup was the highest,but there was no significant difference between 24 h and 36 h subgroup ( P>0.05). (3) There was negative correlation between Bcl-2 and AI (r=-0.753, P<0.001). Conclusion Myocardial apoptosis may be one of mechanisms for myocardial injury in sepsis. Anti-apoptosis intervention may be a promising therapy to improve the prognosis of sepsis. Key words: Sepsis; Rat; Myocardial; Apoptosis; Bcl-2; Caspase-3

ADMINISTRATION OF EXOGENOUS FRACTALKINE, A CX3C CHEMOKINE, IS CAPABLE OF MODULATING INFLAMMATORY RESPONSE IN CECAL LIGATION AND PUNCTURE-INDUCED SEPSIS

Fractalkine (FTK) is a unique member of the CX3C chemokine family by acting through the CX3CR1 receptor. Membrane-bound FTK acts like an adhesion molecule, whereas soluble FTK (sFTK) acts as a classic chemokine ligand. Whether this chemokine plays a role in sepsis is still not clear. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found that FTK levels were elevated in plasma 24 h after CLP. Reverse transcription-polymerase chain reaction results showed that FTK messenger RNA levels were upregulated, whereas CX3CR1 messenger RNA levels were downregulated in lungs after CLP procedure. To study the role of FTK in lung injury during sepsis, we injected exogenous sFTK into the mice before the CLP procedure. We found that plasma FTK levels were further elevated by sFTK. Mice that were injected with FTK had a lower myeloperoxidase activity in lungs compared with the CLP group. Furthermore, macrophage inflammatory protein 2, IL-1beta, and IL-6 levels in lungs were reduced after the injection of FTK. Treatment with sFTK also attenuated lung morphological changes in histological sections. To find out whether sFTK had an effect on leukocyte rolling and adherence, intravital microscope was used. Results showed that sFTK significantly attenuated leukocyte adhesion but had little effect on leukocyte rolling in mesenteric microcirculation. Taken together, our findings suggest that FTK may be a novel chemokine that modulates neutrophil infiltration and chemokine and cytokine production during sepsis.

Immunodesign of experimental sepsis by cecal ligation and puncture

Sepsis remains a prevalent clinical challenge and the underlying pathophysiology is still poorly understood. To investigate the complex molecular mechanisms of sepsis, various animal models have been developed, the most frequently used being the cecal ligation and puncture (CLP) model in rodents. In this model, sepsis originates from a polymicrobial infectious focus within the abdominal cavity, followed by bacterial translocation into the blood compartment, which then triggers a systemic inflammatory response. A requirement of this model is that it is performed with high consistency to obtain reproducible results. Evidence is now emerging that the accompanying inflammatory response varies with the severity grade of sepsis, which is highly dependent on the extent of cecal ligation. In this protocol, we define standardized procedures for inducing sepsis in mice and rats by applying defined severity grades of sepsis through modulation of the position of cecal ligation. The CLP procedure can be performed in as little as 10 min for each animal by an experienced user, with additional time required for subsequent postoperative care and data collection.

Protective Effects of N-Acetylcysteine and β -Glucan Pretreatment on Oxidative Stress in Cecal Ligation and Puncture Model of Sepsis

This study was designed to compare the effect of pretreatment with N-acetylcysteine (NAC) and β -glucan (β GLU) on inflammatory response in a rat model of sepsis. The study was performed in the animal laboratory of the Kahramanmaras Sutcu Imam University, School of Medicine. Forty rats were randomized into four groups (control, sham, NAC, and β GLU). Control and Sham groups received saline or NAC (200 mg/kg, po) in the NAC group and β GLU (50 mg/kg, po) in the βGLU group via intragastric gavage once a day for 10 days and 30 min prior to surgery. Sepsis was induced by cecal ligation and puncture (CLP) in rats. In the NAC, β GLU, and control groups, a laparotomy was performed with the CLP procedure. In the sham group, laparotomy was performed and cecum was manipulated but not ligated or perforated. TNF-α and IL-6 levels were significantly elevated in the control group and decreased in the NAC and β GLU groups. IL-10 levels were significantly increased in the β GLU group (p <. 05). Superoxide dismutase and catalase levels in the liver tissue were significantly increased in the NAC and β GLU groups, whereas superoxide dismutase levels were higher in the β GLU pretreatment group than the NAC pretreatment group (p < 0.05). Malondialdehyde levels in the liver tissue were significantly elevated in the control group and decreased in the NAC and β GLU groups (p <. 05). Prophylactic administration of NAC or β GLU similarly ameliorated sepsis syndrome by reduction of the proinflammatory cytokines and increase of the anti-inflammatory cytokine levels and accession of cellular antioxidants, which protect cells from oxidative stress, thereby recruiting inflammatory cells into tissue.

Neurokinin-1 receptor antagonist treatment protects mice against lung injury in polymicrobial sepsis

Earlier work from our laboratory has suggested a role for the neuropeptide substance P (SP) in inducing lung injury in sepsis. In that study, mice lacking the preprotachykinin-A gene, which encodes for SP, were protected against lung injury in sepsis. To further substantiate the role of SP in sepsis and to study its mechanism, we have evaluated the effect of SR140333, a SP receptor antagonist, on lung injury in sepsis, which was induced in male Swiss mice by cecal ligation and puncture (CLP). Sham-operated animals received the same surgical procedure, except CLP. Vehicle or SR140333 (1 mg/kg, s.c.) was administered to CLP mice 30 min before or 1 h after the CLP. Eight hours after surgery, lung tissue was collected and analyzed for myeloperoxidase (MPO) activity, chemokines, cytokines, and adhesion molecules. The CLP procedure alone caused a significant increase in the lung levels of MIP-2, MCP-1, IL-1beta, IL-6, ICAM-1, E- and P-selectin, and MPO activity when compared with sham-operated mice. SR140333 injected 30 min before or 1 h after CLP significantly attenuated the increased lung MPO activity and levels of MIP-2, MCP-1, IL-1beta, IL-6, ICAM-1, and E- and P-selectin compared with CLP-operated mice injected with the vehicle. Histological evaluation of the lung sections further supported the beneficial effect of SR140333 on lung inflammation. Therefore, SP receptor antagonism can be a potential therapeutic target in polymicrobial sepsis, and this effect is brought about via reduction in leukocyte recruitment.

Anti‐High‐Mobility Group Box Chromosomal Protein 1 Antibodies Improve Survival of Rats with Sepsis

High-mobility group box chromosomal protein 1 (HMGB1) has recently been shown to be an important late mediator of endotoxin shock, intraabdominal sepsis, and acute lung injury, and a promising therapeutic target of severe sepsis. We sought to investigate the effect of antibodies to HMGB1 on severe sepsis in a rat cecal ligation and puncture (CLP) model. Adult male Sprague-Dawley rats underwent CLP and then were randomly divided into two groups: treatment with anti-HMGB1 polyclonal antibodies, and non-immune IgG-treated controls. The serum HMGB1 concentrations were measured at ten time points (preoperatively, and postoperatively at 4, 8, 20, 32, and 48 h and at 3, 4, 5, and 6 days). Hematoxylin-eosin staining, elastica-Masson staining, and immunohistochemical staining for HMGB1 were performed on the cecum and the lung to assess pathological changes 24 h after the CLP procedure. Treatment with anti-HMGB1 antibodies significantly increased survival [55% (anti-HMGB1) vs. 9% (controls); P< 0.01]. The serum HMGB1 concentrations at postoperative hours 20 and 32 of the anti-HMGB1 antibody-treated animals were significantly lower than those of the controls (P < 0.05). Treatment with anti-HMGB1 antibodies markedly diminished the pathological changes and the number of HMGB1-positive cells in the cecum and the lung. The present study demonstrates that anti-HMGB1 antibodies are effective in the treatment of severe sepsis in a rat model, thereby supporting the relevance of HMGB1 eradication therapy for severe sepsis.

Effect of Melatonin on Healing of Colonic Anastomosis in a Rat Model of Peritonitis

Background: The aim of this study is to determine the effect of melatonin on intestinal anastomosis in the presence of peritonitis. Material and Methods: 32 Wistar albino rats were randomized into four groups (n = 8): A (sham), B (control), C (melatonin 5 mg/kg), and D (melatonin 10 mg/kg). In group A, only cecal dissection was carried out. In the other groups, cecal ligation and puncture (CLP) followed cecal dissection in order to induce bacterial peritonitis. 24 h after the previous operation, cecal resection and iliocolic anastomosis were performed in the rats of all groups. In group C (5 mg/kg) and group D (10 mg/kg), melatonin was injected for 5 consecutive days starting after CLP. At the 48th hour of the CLP procedure, blood was drawn via the tail vein for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) analysis, and on the 4th day of the experiment relaparotomy was carried out for bursting pressure (BP) measurements. The intestinal tissue containing the anastomotic line was then snap-frozen in liquid nitrogen and stored at –80°C for determination of tissue levels of malondialdehyde (MDA) and glutathione (GSH). Results: The tissue MDA level, blood TNF-α and IL-6 levels of group B were significantly higher than in the other groups, whereas the BP results and GSH levels of group B were found to be significantly lower than in the other groups. The results of groups C and D are statistically different from those of group B. When we compared the results of groups C and D, we found significantly higher results in terms of BP and GSH levels in group D and also significantly lower results in terms of MDA, blood TNF-α and IL-6 levels in group D. Conclusion: The findings of this experiment suggest that melatonin has a dose-independent positive effect on wound healing of colonic anastomosis.

N-ACETYLCYSTEINE PROTECTS THE RATS AGAINST PHRENIC NERVE DYSFUNCTION IN SEPSIS

This study investigates the association of oxidative stress with the function of the phrenic nerve and inquires whether N-acetylcysteine (NAC) may counteract the possible detrimental effects. Thirty rats were divided into three groups: sham, cecal ligation and puncture (CLP), and CLP plus NAC treatment. Sepsis was produced by the CLP procedure. NAC was administered at 70 mg/day for 7 days. Electrophysiology was evaluated by the needle electromyography of the diaphragm and phrenic nerve conduction study. Oxidative stress was evaluated by malondialdehyde (MDA), nitrite/nitrate (NN), and reduced-glutathione (ReGSH) levels and myeloperoxidase (MPO) and catalase (CAT) activities in the phrenic nerve. In the CLP group, ReGSH and CAT were decreased (P = 0.0001, P = 0.07, respectively); and MDA, MPO, and NN were increased (P = 0.02, P = 0.0001, P = 0.043, respectively), compared with the sham group. NAC administration increased the ReGSH (P = 0.036) and decreased the MDA, MPO, and NN (P = 0.008, P = 0.01, P = 0.032, respectively), compared with the CLP group. In the CLP group, electrophysiology revealed reductions in the number of motor unit action potentials (P = 0.0001) and prolongations in the latency of the compound nerve action potential (P = 0.0001), indicating phrenic nerve neuropathy. NAC administration significantly ameliorated these electrophysiological alterations (P = 0.011, P = 0.0001, respectively), compared with the CLP group. The present results showed that intraabdominal sepsis is closely associated with phrenic nerve neuropathy. In addition, NAC administration protects the rats against the detrimental events of sepsis.

Effect of Alpha-Tocopherol on the Expression of Hepatic Vascular Stress Genes in Response to Sepsis

Sepsis is the leading cause of death in critically ill surgical patients. Septic hepatic dysfunction, an important determinant of outcome, although poorly understood, includes inappropriate expression of vasoregulatory genes. In this study the effect of alpha-tocopherol was determined on the expression of hepatic vascular stress genes in response to sepsis. Rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Rats received either vehicle or alpha-tocopherol (AT, 15 mg/kg), intraperitoneally injected for 3 d prior to CLP procedure. Serum aminotransferase activities and hepatic lipid peroxides levels markedly increased 24 h after CLP, and this rise was attenuated by AT treatment. The hepatic concentrations of reduced glutathione decreased in CLP animals, which was inhibited by AT. CLP significantly increased mRNA levels of endothelin (ET)-1 and ETB receptor in livers, which was not prevented by AT treatment. There were no significant changes in ETA mRNA expression among any of the experimental groups. There were significant increases in the mRNA expression of inducible nitric oxide synthase and heme oxygenase-1 in livers of CLP animals, and this was prevented by AT treatment. The expression of tumor necrosis factor-α and cyclooxygenase-2 mRNA increased 4.9-fold and 4.4-fold, respectively, in livers of CLP animals. This increase was attenuated by AT treatment. Our data suggest that sepsis induces an imbalance in hepatic vasoregulatory gene expression and that AT ameliorates altered expression of vasodilators through its free radical scavenging activity.

The Effect of Preventive Use of Alanyl-Glutamine on Diaphragm Muscle Function in Cecal Ligation and Puncture-Induced Sepsis Model

Low muscle glutamine levels during sepsis are associated with reduced protein synthesis and elevated protein breakdown, in particular myofibrillar protein breakdown. Thus, in a cecal ligation and puncture (CLP)-induced sepsis model in the rat, we hypothesized that glutamine pretreatment would protect the diaphragm muscle function. Eighty-four male Wistar rats weighing between 180 g and 200 g received standard amino acid solution 1.2 g kg(-1) per day intraperitoneally (IP) or standard amino acid solution 1.2 g kg(-1) per day plus alanyl-glutamine (GLN) 0.25 g kg(-1) per day (IP) during the first 6 days of the experiment. On the seventh day, CLP or sham procedures were applied. The sham and CLP groups were equally divided into 3 subgroups according to the termination of the experiment, which took place at either the 24th hour, 48th hour, or 72nd hour. After the compound muscle action potentials (CMAP) were recorded from the diaphragms of the rats at these selected times, they were decapitated under ketamine/xylazine anesthesia, and diaphragms were harvested for biochemical and histopathological examination. The mean area and amplitude of CMAP were significantly larger in sham+GLN groups when compared with CLP and CLP+GLN groups at all times (p < .05). Diaphragm Ca+2 -ATPase levels were found to be significantly decreased in CLP group at all times compared to sham groups (p < .05). Diaphragm reduced glutathione levels were significantly higher in sham+GLN groups when compared with CLP and CLP+GLN groups at all times (p < .05). In histopathologic assessment, moderate neutrophil infiltration, which was observed in CLP48, was significantly reduced with alanyl-glutamine supplementation in CLP+GLN48 group (p < .05). This study showed that glutamine pretreatment did not improve diaphragm muscle function, but prevented the biochemical and histopathological changes in diaphragmatic muscle in CLP-induced sepsis. However, further studies are needed to clarify whether a higher dose of glutamine supplementation might protect the diaphragmatic muscle functions.