Febrile response induced by cecal ligation and puncture (CLP) in rats: involvement of prostaglandin E-2 and cytokines

The purpose of the present study was to better understand the events involved in the febrile response induced by cecal ligation and puncture (CLP), a complex infectious process. To this end, we conducted in vivo experiments in rats examining (1) fever development, (2) bacterial number in the infection focus and in blood, (3) peripheral and hypothalamic synthesis of cytokines, (4) hypothalamic and cerebrospinal fluid (CSF) synthesis of prostaglandin E-2 (PGE(2)), (5) the effect of anti-IL-6 antibody on fever, and (6) the effect of celecoxib on fever and hypothalamic synthesis of PGE(2) after CLP induction. We found that CLP promotes fever and animal death depending on the number of punctures. The peak of CLP-induced fever overlapped with the maximal increase in the number of bacteria in the infectious focus and blood, which occurred at 6 and 12 h. The peak of the febrile response also coincided with increased amounts of interleukin (IL)-1 beta, IL-6 and IL-10 in the peritoneal exudate and serum; IL-6 in the hypothalamus and PGE(2) in the CSF and predominantly in the hypothalamus. Moreover, intracerebroventricularly injected anti-IL-6 antibody reduced the febrile response while celecoxib reduced the fever and PGE(2) amount in the hypothalamus induced by CLP. Tumor necrosis factor (TNF)-alpha peaked at 3 h at all sites studied. Conversely, IL-10 concentration decreased in the hypothalamus. These findings show that the peak of CLP-induced fever is accompanied by an increase of bacteria in peritoneal fluid (local infection) and blood; local synthesis of pyrogenic (IL-1 beta, IL-6) and antipyretic (IL-10) cytokines and central production of IL-6 and PGE(2), suggesting that these last are the central mediators of this response.

Alterations in pulmonary structure by elastase administration in a model of emphysema in mice is associated with functional disturbances

Several experimental studies of pulmonary emphysema using animal models have been described in the literature. However, only a few of these studies have focused on the assessment of ergometric function as a non-invasive technique to validate the methodology used for induction of experimental emphysema. Additionally, functional assessments of emphysema are rarely correlated with morphological pulmonary abnormalities caused by induced emphysema. The present study aimed to evaluate the effects of elastase administered by tracheal puncture on pulmonary parenchyma and their corresponding functional impairment. This was evaluated by measuring exercise capacity in C57Bl/6 mice in order to establish a reproducible and safe methodology of inducing experimental emphysema. Thirty six mice underwent ergometric tests before and 28 days after elastase administration. Pancreatic porcine elastase solution was administered by tracheal puncture, which resulted in a significantly decreased exercise capacity, shown by a shorter distance run (-30.5%) and a lower mean velocity (-15%), as well as in failure to increase the elimination of carbon dioxide. The mean linear intercept increased significantly by 50% in tracheal elastase administration. In conclusion, application of elastase by tracheal function in C57Bl/6 induces emphysema, as validated by morphometric analyses, and resulted in a significantly lower exercise capacity, while resulting in a low mortality rate. (C) 2011 Sociedade Portuguesa de Pneumologia. Published by Elsevier Espana, S.L. All rights reserved.

Bone marrow cells of swine: collection and separation

Bone marrow is a source of stem cells for greater and easier access, which is widely studied as a provider of hematopoietic and mesenchymal cells for various purposes, mainly therapeutic by the advances in research involving cell therapy. The swine is an animal species commonly used in the pursuit of development of experimental models. Thus, this study aimed to standardize protocol for collection and separation of bone marrow in swines, since this species is widely used as experimental models for various diseases. Twelve animals were used, which underwent bone marrow puncture with access from the iliac crest and cell separation by density gradient followed by a viability test with an average of 98% of viable cells. Given our results, we can ensure the swine as an excellent model for obtaining and isolation of mononuclear cells from bone marrow, stimulating several studies addressing the field of cell therapy. Microsc. Res. Tech., 2012. (C) 2012 Wiley Periodicals, Inc.

Minimal residual disease in cerebrospinal fluid at diagnosis: a more intensive treatment protocol was able to eliminate the adverse prognosis in children with acute lymphoblastic leukemia

We analyzed cerebrospinal fluid (CSF) samples from 65 consecutive children with acute lymphoblastic leukemia (ALL) treated according to two different treatment protocols (GBTLI-ALL-93 and -99) with no puncture accident for minimal residual disease (MRD) in the central nervous system (CNS). Minimal residual disease was detected by polymerase chain reaction (PCR) with homo/heteroduplex analysis using consensus primers to IgH and TCR genes. MRD in the CSF at diagnosis was detected by PCR in 46.8% of children with no puncture accident or morphological involvement. In patients treated with GBTLI-ALL-93 a significantly lower 5-year event-free survival (EFS) was demonstrated for those with CSF involvement, in univariate (p = 0.01) and multivariate (p = 0.04) analysis. This observation was not true for patients treated with the more intensive protocol GBTLI-ALL-99 (p = 0.81). These findings suggest that MRD detection in the CSF is a common event in children with ALL. Treatment intensification provided by the GBTLI-ALL-99 apparently overcomes the detrimental effect of CNS minimal residual disease at diagnosis.

Sepsis-Induced Acute Lung Injury (ALI) Is Milder in Diabetic Rats and Correlates with Impaired NFkB Activation

Acute lung injury (ALI) develops in response to a direct insult to the lung or secondarily to a systemic inflammatory response, such as sepsis. There is clinical evidence that the incidence and severity of ALI induced by direct insult are lower in diabetics. In the present study we investigated whether the same occurs in ALI secondarily to sepsis and the molecular mechanisms involved. Diabetes was induced in male Wistar rats by alloxan and sepsis by caecal ligation and puncture surgery (CLP). Six hours later, the lungs were examined for oedema and cell infiltration in bronchoalveolar lavage. Alveolar macrophages (AMs) were cultured in vitro for analysis of I kappa B and p65 subunit of NF kappa B phosphorylation and MyD88 and SOCS-1 mRNA. Diabetic rats were more susceptible to sepsis than non-diabetics. In non-diabetic rats, the lung presented oedema, leukocyte infiltration and increased COX2 expression. In diabetic rats these inflammatory events were significantly less intense. To understand why diabetic rats despite being more susceptible to sepsis develop milder ALI, we examined the NF kappa B activation in AMs of animals with sepsis. Whereas in non-diabetic rats the phosphorylation of I kappa B and p65 subunit occurred after 6 h of sepsis induction, this did not occur in diabetics. Moreover, in AMs from diabetic rats the expression of MyD88 mRNA was lower and that of SOCS-1 mRNA was increased compared with AMs from non-diabetic rats. These results show that ALI secondary to sepsis is milder in diabetic rats and this correlates with impaired activation of NF kappa B, increased SOCS-1 and decreased MyD88 mRNA.

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

Maternal factors regulating preterm infants' responses to pain and stress while in maternal kangaroo care

The relationship between maternal factors and the response of preterm infants to pain and stress experienced during heel puncture while in maternal kangaroo care was investigated. This descriptive study included 42 mothers and their preterm infants cared for in a neonatal unit. Data were collected in the baseline, procedure, and recovery phases. We measured the neonates' facial actions, sleep and wake states, crying, salivary cortisol levels, and heart rate, in addition to the mothers' behavior, salivary cortisol levels, and mental condition. The influence of the maternal explanatory variables on the neonatal response variables were verified through bivariate analysis, ANOVA, and multiple regression. The mothers' behavior and depression and/or anxiety did not affect the neonates' responses to pain and stress, though the mothers' levels of salivary cortisol before the procedure explained the variance in the neonates' levels of salivary cortisol after the procedure (p=0.036). Additionally, the mothers' baseline levels of salivary cortisol along with the neonates' age explained the variance in the neonates' heart rate (p=0.001). The ability of mothers to regulate their own stress contributed to the infants' responses to pain and stress.

N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation

Campos R, Shimizu MH, Volpini RA, de Bragan a AC, Andrade L, Lopes FD, Olivo C, Canale D, Seguro AC. N-acetylcysteine prevents pulmonary edema and acute kidney injury in rats with sepsis submitted to mechanical ventilation. Am J Physiol Lung Cell Mol Physiol 302: L640-L650, 2012. First published January 20, 2012; doi: 10.1152/ajplung.00097.2011.-Sepsis is a common cause of acute kidney injury (AKI) and acute lung injury. Oxidative stress plays as important role in such injury. The aim of this study was to evaluate the effects that the potent antioxidant N-acetylcysteine (NAC) has on renal and pulmonary function in rats with sepsis. Rats, treated or not with NAC (4.8 g/l in drinking water), underwent cecal ligation and puncture (CLP) 2 days after the initiation of NAC treatment, which was maintained throughout the study. At 24 h post-CLP, renal and pulmonary function were studied in four groups: control, control + NAC, CLP, and CLP + NAC. All animals were submitted to low-tidal-volume mechanical ventilation. We evaluated respiratory mechanics, the sodium cotransporters Na-K-2Cl (NKCC1) and the alpha-subunit of the epithelial sodium channel (alpha-ENaC), polymorphonuclear neutrophils, the edema index, oxidative stress (plasma thiobarbituric acid reactive substances and lung tissue 8-isoprostane), and glomerular filtration rate. The CLP rats developed AKI, which was ameliorated in the CLP + NAC rats. Sepsis-induced alterations in respiratory mechanics were also ameliorated by NAC. Edema indexes were lower in the CLP + NAC group, as was the wet-to-dry lung weight ratio. In CLP + NAC rats, alpha-ENaC expression was upregulated, whereas that of NKCC1 was downregulated, although the difference was not significant. In the CLP + NAC group, oxidative stress was significantly lower and survival rates were significantly higher than in the CLP group. The protective effects of NAC (against kidney and lung injury) are likely attributable to the decrease in oxidative stress, suggesting that NAC can be useful in the treatment of sepsis.

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-kappa B and upregulating endothelial nitric oxide synthase

de Souza ACCP, Volpini RA, Shimizu MH, Sanches TR, Camara NOS, Semedo P, Rodrigues CE, Seguro AC, Andrade L. Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting nuclear factor-kappa B and upregulating endothelial nitric oxide synthase. Am J Physiol Renal Physiol 302: F1045-F1054, 2012. First published January 11, 2012; doi:10.1152/ajprenal.00148.2011.-The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-kappa B activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP + EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-L-arginine methyl ester (L-NAME) simultaneously with EPO administration (CLP + EPO + L-NAME). A fifth group (CLP + EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP + EPO rats presented significantly higher inulin clearance than did CLP and CLP + EPO + L-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP + EPO rats; and inulin clearance was significantly higher in CLP + EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP + EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-alpha activation, NF-kappa B activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-kappa B downregulation.

Toll-like receptor 9 activation in neutrophils impairs chemotaxis and reduces sepsis outcome

Objectives: To investigate the role of toll-like receptor 9 on sepsis-induced failure of neutrophil recruitment to the site of infection. Design: Prospective experimental study. Setting: University research laboratory. Interventions: Model of polymicrobial sepsis induced by cecal ligation and puncture in wild-type and toll-like receptor 9-deficient mice. Measurements and Main Results: Toll-like receptor 9-deficient mice with cecal ligation and puncture-induced severe sepsis did not demonstrate failure of neutrophil migration and consequently had a low systemic inflammatory response and a high survival rate. Upon investigating the mechanism by which toll-like receptor 9-deficiency prevents the failure of neutrophil migration, it was found that neutrophils derived from toll-like receptor 9-deficient mice with cecal ligation and puncture induced severe sepsis expressed high levels of chemokine C-X-C motif receptor 2 (CXCR2) and had reduced induction of G-protein-coupled receptor kinase 2. Conclusions: These findings suggest that the poor outcome of severe sepsis is associated with toll-like receptor 9 activation in neutrophils, which triggers G-protein-coupled receptor kinase 2 expression and CXCR2 downregulation. These events account for the reduction of neutrophil migration to the site of infection, with consequent spreading of the infection, onset of the systemic inflammatory response, and a decrease in survival. (Crit Care Med 2012; 40:2631-2637)

Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury

de Araujo CC, Silva JD, Samary CS, Guimaraes IH, Marques PS, Oliveira GP, do Carmo LGRR, Goldenberg RC, Bakker-Abreu I, Diaz BL, Rocha NN, Capelozzi VL, Pelosi P, Rocco PRM. Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury. J Appl Physiol 112: 1206-1214, 2012. First published January 19, 2012; doi:10.1152/japplphysiol.01061.2011.-Physical activity modulates inflammation and immune response in both normal and pathologic conditions. We investigated whether regular and moderate exercise before the induction of experimental sepsis reduces the risk of lung and distal organ injury and survival. One hundred twenty-four BALB/c mice were randomly assigned to two groups: sedentary (S) and trained (T). Animals in T group ran on a motorized treadmill, at moderate intensity, 5% grade, 30 min/day, 3 times a week for 8 wk. Cardiac adaptation to exercise was evaluated using echocardiography. Systolic volume and left ventricular mass were increased in T compared with S group. Both T and S groups were further randomized either to sepsis induced by cecal ligation and puncture surgery (CLP) or sham operation (control). After 24 h, lung mechanics and histology, the degree of cell apoptosis in lung, heart, kidney, liver, and small intestine villi, and interleukin (IL)-6, KC (IL-8 murine functional homolog), IL-1 beta, IL-10, and number of cells in bronchoalveolar lavage (BALF) and peritoneal lavage (PLF) fluids as well as plasma were measured. In CLP, T compared with S groups showed: 1) improvement in survival; 2) reduced lung static elastance, alveolar collapse, collagen and elastic fiber content, number of neutrophils in BALF, PLF, and plasma, as well as lung and distal organ cell apoptosis; and 3) increased IL-10 in BALF and plasma, with reduced IL-6, KC, and IL-1 beta in PLF. In conclusion, regular and moderate exercise before the induction of sepsis reduced the risk of lung and distal organ damage, thus increasing survival.

Effect of a video on developing skills in undergraduate nursing students for the management of totally implantable central venous access ports

Background: Handling Totally Implantable Access Ports (TIAP) is a nursing procedure that requires skill and knowledge to avoid adverse events. No studies addressing this procedure with undergraduate students were identified prior to this study. Communication technologies, such as videos, have been increasingly adopted in the teaching of nursing and have contributed to the acquisition of competencies for clinical performance. Objective: To evaluate the effect of a video on the puncture and heparinization of TIAP in the development of cognitive and technical competencies of undergraduate nursing students. Method: Quasi-experimental study with a pretest-posttest design. Results: 24 individuals participated in the study. Anxiety scores were kept at levels 1 and 2 in the pretest and posttest. In relation to cognitive knowledge concerning the procedure, the proportion of correct answers in the pretest was 0.14 (SD=0.12) and 0.90 in the posttest (SD=0.05). After watching the video, the average score obtained by the participants in the mock session was 27.20. Conclusion: The use of an educational video with a simulation of puncture and heparinization of TIAP proved to be a strategy that increased both cognitive and technical knowledge. This strategy is viable in the teaching-learning process and is useful as a support tool for professors and for the development of undergraduate nursing students. (C) 2011 Elsevier Ltd. All rights reserved.

Revisiting a medical case of "stinging" in the human oral cavity caused by ingestion of raw squid (Cephalopoda: Teuthida): new data on the functioning of squid's spermatophores

Male squid produce intricate spermatophores that, when transferred to the female, undergo the spermatophoric reaction, a complex process of evagination that leads to the attachment of the spermatangium, that is, the everted spermatophore containing the sperm mass. While this process is still not completely understood, the medical literature includes several reports of "oral stinging" (i.e., punctured wounds in the human oral cavity) following consumption of raw male squid, which contains undischarged spermatophores able to inflict such wounds. Here, we revisit a recent medical report of oral stinging by Shiraki et al. (Pathol Int 61:749-751, 2011), providing an in-depth reanalysis of their histological biopsies and revealing vital information on the functioning of squid spermatophores. The morphology of the spermatangia attached within the oral cavity is similar to the condition found in spermatangia naturally attached to female squids. The spermatangia were able to superficially puncture the superficial layers of the oral stratified squamous epithelium, and numerous, minute stellate particles from the squid spermatophore were found adhered to the oral epithelium. These findings corroborate previous hypotheses on the functioning of squid spermatophores, namely that spermatophore attachment generally involves tissue scarification, and that stellate particles play a vital role in the attachment process. Moreover, spermatophore attachment is confirmed to be autonomous (i.e., performed by the spermatophore itself) in another squid species (possibly a loliginid), and the results strongly indicate that the attachment mechanism is not dependent upon a specialized epithelium, nor a mate's specific chemical stimulus. From the pathological point of view, the best prophylactic measure at present is the removal of the internal organs of the raw squid prior to its consumption.

Does the neonatal clinical risk for illness severity influence pain reactivity and recovery in preterm infants?

Background: The biobehavioural pain reactivity and recovery of preterm infants in the neonatal period may reflect the capacity of the central nervous system to regulate neurobiological development. Objective: The aim of the present study was to analyse the influence of the neonatal clinical risk for illness severity on biobehavioural pain reactivity in preterm infants. Methods: Fifty-two preterm infants were allocated into two groups according to neonatal severity of illness, as measured by the Clinical Risk Index for Babies (CRIB). The low clinical risk (LCr) group included 30 neonates with CRIB scores <4, and the high clinical risk (HCr) group included 22 neonates with CRIB scores >= 4. Pain reactivity was assessed during a blood collection, which was divided into five phases (baseline, antisepsis, puncture, recovery-dressing and recovery-resting). Behavioral pain reactivity was measured using the scores, and magnitude of responses in Neonatal Facial Coding System (NFCS) and Sleep-Wake States Scale (SWS). The heart rate was continuously recorded. Results: The HCr demonstrated a higher magnitude of response on the SWS score from the baseline to the puncture phase than the LCr. Also, the HCr exhibited a higher mean heart rate and minimum heart rate than the LCr in the recovery-resting phase. In addition, the HCr exhibited a higher minimum heart rate from the baseline to the recovery-resting phase than the LCr. Conclusion: The infants exhibiting a high neonatal clinical risk showed high arousal during the puncture procedure and higher physiological reactivity in the recovery phase.

Physical properties of edible films based on bovine myofibril proteins

Myofibril proteins have excellent filmogenic properties. The objective of this article was to study the effect of the thermal treatment, of the pH and of the plasticizer concentration (Cp) of the filmogenic solution (FS), using over some physical properties of edible films, using a surface and response methodology (SRM). Films were made of lyophilized myofibril proteins (LMP) extracted from bovine muscle, employing the technique of solubility obtained from diluted saline solutions. The films were elaborated from FS containing 1 g of LMP/100g of FS and from Cp of 50 g to 79 g of glycerin/100 g of LMP. The LMP was dispersed in water under moderate agitation, and the pH was kept at 2.5-3.5 with the use of acetic acid. The FS were submitted to thermal treatment at different temperatures for 45 minutes. Films were dried in ventilated oven at 37 degrees C/18hr, conditioned at 75% of relative humidity at 25 degrees C/48 hr before analysis of: mechanical properties by puncture test; apparent opacity by spectrophotometer; solubility by immersion in water; and water vapor permeability by the gravimetric method. In general, films showed good appearance, translucent, easily handled and touchable, except for the films formed with pH 2.5 and at a low temperature (35 degrees C), with a medium thickness of 0.400 +/- 0.005 mm. The pH of the FS significantly affected all the physical properties under study. The temperature of the thermal treatment of the FS greatly affected the force at the rupture, solubility and water vapor permeability. This treatment can promote intermolecular interactions through the formation of disulphide bonds; however a very intense treatment can reverse this effect by irreversible structural alterations in the proteins. The glycerol concentration affected considerably all the properties under study, with the exception of the apparent opacity. Plasticizer increases the mobility of macromolecules with consequences in all physical properties.