Partial Replacement of omega-6 Fatty Acids With Medium-Chain Triglycerides, but Not Olive Oil, Improves Colon Cytokine Response and Damage in Experimental Colitis

Background: Soybean oil is rich in omega-6 fatty acids, which are associated with higher incidence and more severe cases of inflammatory bowel diseases. The authors evaluated whether partial replacement of soybean oil by medium-chain triglycerides (MCTs) or olive oil influenced the incidence and severity of experimental ulcerative colitis by using different parenteral lipid emulsions (LEs). Methods: Wistar rats (n = 40) were randomized to receive parenteral infusion of the following LE: 100% soybean oil (SO), 50% MCT mixed with 50% soybean oil (MCT/SO), 80% olive oil mixed with 20% soybean oil (OO/SO), or saline (CC). After 72 hours of infusion, acetic acid experimental colitis was induced. After 24 hours, colon histology and cytokine expression were analyzed. Results: SO was not significantly associated with overall tissue damage. MCT/SO was not associated with necrosis (P < .005), whereas OO/SO had higher frequencies of ulcer and necrosis (P < .005). SO was associated with increased expression of interferon-gamma (P = .005) and OO/SO with increased interleukin (IL)-6 and decreased tumor necrosis factor-alpha expression (P < .05). MCT/SO appeared to decrease IL-1 (P < .05) and increase IL-4 (P < .001) expression. Conclusions: Parenteral SO with high concentration of omega-6 fatty acids was not associated with greater tissue damage in experimental colitis. SO partial replacement with MCT/SO decreased the frequency of histological necrosis and favorably modulated cytokine expression in the colon; however, replacement with OO/SO had unfavorable effects. (JPEN J Parenter Enteral Nutr. 2012; 36: 442-448)

Improvement in ARDS experimental model installation: Low mortality rate and maintenance of hemodynamic stability

Introduction: Many experimental models using lung lavage have been developed for the study of acute respiratory distress syndrome (ARDS). The original technique has been modified by many authors, resulting in difficulties with reproducibility. There is insufficient detail on the lung injury models used, including hemodynamic stability during animal preparation and drawbacks encountered such as mortality. The authors studied the effects of the pulmonary recruitment and the use of fixed tidal volume (Vt) or fixed inspiratory pressure in the experimental ARDS model installation. Methods: Adult rabbits were submitted to repeated lung lavages with 30 ml/kg warm saline until the ARDS definition (PaO2/FiO(2) <= 100) was reached. The animals were divided into three groups, according to the technique used for mechanical ventilation: 1) fixed Vt of 10 ml/kg; 2) fixed inspiratory pressure (IP) with a tidal volume of 10 ml/kg prior to the first lung lavage; and 3) fixed Vt of 10 ml/kg with pulmonary recruitment before the first lavage. Results: The use of alveolar recruitment maneuvers, and the use of a fixed Vt or IP between the lung lavages did not change the number of lung lavages necessary to obtain the experimental model of ARDS or the hemodynamic stability of the animals during the procedure. A trend was observed toward an increased mortality rate with the recruitment maneuver and with the use of a fixed IP. Discussion: There were no differences between the three study groups, with no disadvantage in method of lung recruitment, either fixed tidal volume or fixed inspiratory pressure, regarding the number of lung lavages necessary to obtain the ARDS animal model. Furthermore, the three different procedures resulted in good hemodynamic stability of the animals, and low mortality rate. (C) 2012 Elsevier Inc. All rights reserved.

Cardiometabolic benefits of exercise training in an experimental model of metabolic syndrome and menopause

Objective: The aim of this study was to investigate the cardiometabolic effects of exercise training in ovariectomized hypertensive rats both submitted and not submitted to fructose overload. Methods: Spontaneously hypertensive ovariectomized rats were divided into sedentary and trained (THO) groups submitted to normal chow and sedentary and trained groups submitted to fructose overload (100 g/L in drinking water for 19 wk). Exercise training was performed on a treadmill (8 wk). Arterial pressure (AP) was directly recorded. Cardiovascular autonomic control was evaluated through pharmacological blockade (atropine and propranolol) and in the time and frequency domains by spectral analysis. Results: The THO group presented reduced AP (approximately 16 mm Hg) and enhanced cardiac vagal tonus (approximately 49%) and baroreflex sensitivity (approximately 43%) compared with the sedentary hypertensive ovariectomized group. Exercise training attenuated metabolic impairment, resting tachycardia, cardiac and vascular sympathetic increases, and baroreflex sensitivity decrease induced by fructose overload in hypertensive rats. However, the trained hypertensive ovariectomized group submitted to fructose overload presented higher AP (approximately 32 mm Hg), associated with baroreflex sensitivity (approximately 69%) and parasympathetic dysfunctions compared with the THO group. Conclusions: These data suggest that the metabolic disorders in hypertensive rats after ovarian hormone deprivation could blunt and/or attenuate some exercise training benefits.

Evaluation of the detection and quantification limits in electroanalysis using two popular methods: application in the case study of paraquat determination

In this work, the reduction reaction of paraquat herbicide was used to obtain analytical signals using electrochemical techniques of differential pulse voltammetry, square wave voltammetry and multiple square wave voltammetry. Analytes were prepared with laboratory purified water and natural water samples (from Mogi-Guacu River, SP). The electrochemical techniques were applied to 1.0 mol L-1 Na2SO4 solutions, at pH 5.5, and containing different concentrations of paraquat, in the range of 1 to 10 mu mol L-1, using a gold ultramicroelectrode. 5 replicate experiments were conducted and in each the mean value for peak currents obtained -0.70 V vs. Ag/AgCl yielded excellent linear relationships with pesticide concentrations. The slope values for the calibration plots (method sensitivity) were 4.06 x 10(-3), 1.07 x 10(-2) and 2.95 x 10(-2) A mol(-1) L for purified water by differential pulse voltammetry, square wave voltammetry and multiple square wave voltammetry, respectively. For river water samples, the slope values were 2.60 x 10(-3), 1.06 x 10(-2) and 3.35 x 10(-2) A mol(-1) L, respectively, showing a small interference from the natural matrix components in paraquat determinations. The detection limits for paraquat determinations were calculated by two distinct methodologies, i.e., as proposed by IUPAC and a statistical method. The values obtained with multiple square waves voltammetry were 0.002 and 0.12 mu mol L-1, respectively, for pure water electrolytes. The detection limit from IUPAC recommendations, when inserted in the calibration curve equation, an analytical signal (oxidation current) is smaller than the one experimentally observed for the blank solution under the same experimental conditions. This is inconsistent with the definition of detection limit, thus the IUPAC methodology requires further discussion. The same conclusion can be drawn by the analyses of detection limits obtained with the other techniques studied.

Effect of topical clay application on the synthesis of collagen in skin: an experimental study

Background. Clay is often used in cosmetic treatments, although little is known about its action. Aim. To evaluate the effect of topical clay application on the histoarchitecture of collagen fibres in rat skin. Methods. Animals received a daily application of clay and retinoic acid (RA) 0.025% to the dorsal skin over 7 and 14 days, under vaporization at 37 degrees C for 40 min. Control skin was not vaporized. Samples from each region were excised, and stained with picrosirius red for collagen evaluation. Results. Seven days after clay treatment, an increase in the number of collagen fibres was observed in treated skin compared with control skin (51.74 +/- 1.28 vs. 43.39 +/- 1.79%, respectively, P < 0.01), whereas RA did not alter the collagen level (45.66 +/- 1.10%). Clay application over 14 days did not induce a further increase in skin collagen, whereas treatment with RA did (58.07 +/- 1.59%; P = 0.001 vs. control). Conclusion. Clay application promotes an increase in the number of collagen fibres, which may account for its beneficial effects.

Effect of Colchicine on Myocardial Injury Induced by Trypanosoma cruzi in Experimental Chagas Disease

Background: The hallmark of Chagas disease (CD) is multifocal myocarditis and extensive fibrosis. We investigated the potential effect of colchicine on myocardial remodeling in experimental CD. Methods and Results: One hundred Syrian hamsters were randomly divided into noninfected untreated control (CG), noninfected control treated with colchicine (COLG 0.4 mg kg(-1) d(-1) by gavage), infected (IG), and infected treated with colchicine (ICOLG, 0.4 mg kg(-1) d(-1)) groups. The interstitial collagen volume fraction (ICVF) was evaluated by videomorphometry with picrosirius red staining. The gelatinolytic activities of matrix metalloproteinase (MMP) 2 were examined with the use of zymography. Myocarditis was described according to the Dallas criteria. Statistical comparisons were performed with parametric analysis of variance and Tukey test. ICVF (%) accumulation was attenuated in infected colchicine-treated animals in the left (CG 0.81 +/- 0.13, COLG 0.85 +/- 0.13, IG: 1.35 +/- 0.31,* ICOLG 1.06 +/- 0.19; *P < .05 compared with ICOLG) and right ventricles (CG 1.4 +/- 0.36, COLG 1.26 +/- 0.14, IG 1.97 +/- 0.058,* ICOLG: 1.52 +/- 0.23; *P < .05 compared with ICOLG). A significant increase in MMP-2 enzymatic activity (UA) was observed in ICOLG (17,432.8*) compared with GC (3731.6), COLG (2,792.6), and IG (4,286.3; *P < .001). In IG, 66% of animals had myocarditis compared with only 49% in ICOLG. Conclusions: Colchicine had a protective effect on myocardium, indicated by decreased interstitial myocardial fibrosis, increased intensity of MMP-2, and attenuated myocardial inflammation. (J Cardiac Fail 2012;18:654-659)

Nitric Oxide Synthase in Heart and Thoracic Aorta After Liver Ischemia and Reperfusion Injury: An Experimental Study in Rats

Objectives: We tested the effects of liver reperfusion in the immunohistochemical expression of nitric oxide synthase on the thoracic aorta and the heart. Materials and Methods: We randomized 24 male Wistar rats into 3 groups: (1) control; (2) R2 group, with 60 minutes of partial (70%) liver ischemia and 2 hours of global liver reperfusion; (3) and R6 group, with 60 minutes of partial liver ischemia and 6 hours of global liver reperfusion. Results: In the heart, there was little, diffuse immunohistochemical endothelial staining; immunohistochemical inducible nitric oxide synthase staining was expressed in the adventitia layer of intramyocardial vessels in both cases, with a time-dependent but not statistically significant increase. In the thoracic aorta, a time-dependent decrease in endothelial nitric oxide synthase expression in the muscular layer after reperfusion, which was statistically significant in R6 versus the control. Positive immunostaining for inducible nitric oxide synthase was seen in the muscular and endothelial layers, and this varied from moderate in the control group, to light in the endothelium in groups R2 and R6. Conclusions: We observed changes that may be implicated in heart injury and impairment of aortal tone after liver ischemia and reperfusion injury.

Diametral tensile strength and film thickness of an experimental dental luting agent derived from castor oil

The need to develop new dental luting agents in order to improve the success of treatments has greatly motivated research. Objective: The aim of this study was to evaluate the diametral tensile strength (DTS) and film thickness (FT) of an experimental dental luting agent derived from castor oil (COP) with or without addition of different quantities of filler (calcium carbonate - CaCO3). Material and Methods: Eighty specimens were manufactured (DTS N=40; FT N=40) and divided into 4 groups: Pure COP; COP 10%; COP 50% and zinc phosphate (control). The cements were mixed according to the manufacturers' recommendations and submitted to the tests. The DTS test was performed in the MTS 810 testing machine (10 KN, 0.5 mm/min). For FT test, the cements were sandwiched between two glass plates (2 cm(2)) and a load of 15 kg was applied vertically on the top of the specimen for 10 min. The data were analyzed by means of one-way ANOVA and Tukey's test (alpha=0.05). Results: The values of DTS (MPa) were: Pure COP- 10.94 +/- 1.30; COP 10%- 30.06 +/- 0.64; COP 50%- 29.87 +/- 0.27; zinc phosphate- 4.88 +/- 0.96. The values of FT (pm) were: Pure COP- 31.09 +/- 3.16; COP 10%- 17.05 +/- 4.83; COP 50%- 13.03 +/- 4.83; Zinc Phosphate- 20.00 +/- 0.12. One-way ANOVA showed statistically significant differences among the groups (DTS - p=1.01E-40; FT - p=2.4E-10). Conclusion: The experimental dental luting agent with 50% of filler showed the best diametral tensile strength and film thickness.

Comparison of two experimental models of pulmonary hypertension

Objective: To compare two models of pulmonary hypertension (monocrotaline and monocrotaline+pneumonectomy) regarding hemodynamic severity, structure of pulmonary arteries, inflammatory markers (IL-1 and PDGF), and 45-day survival. Methods: We used 80 Sprague-Dawley rats in two study protocols: structural analysis; and survival analysis. The rats were divided into four groups: control; monocrotaline (M), pneumonectomy (P), and monocrotaline+pneumonectomy (M+P). In the structural analysis protocol, 40 rats (10/group) were catheterized for the determination of hemodynamic variables, followed by euthanasia for the removal of heart and lung tissue. The right ventricle (RV) was dissected from the interventricular septum (IS), and the ratio between RV weight and the weight of the left ventricle (LV) plus IS (RV/LV+IS) was taken as the index of RV hypertrophy. In lung tissues, we performed histological analyses, as well as using ELISA to determine IL-1 and PDGF levels. In the survival protocol, 40 animals (10/group) were followed for 45 days. Results: The M and M+P rats developed pulmonary hypertension, whereas the control and P rats did not. The RV/LV+IS ratio was significantly higher in M+P rats than in M rats, as well as being significantly higher in M and M+P rats than in control and P rats. There were no significant differences between the M and M+P rats regarding the area of the medial layer of the pulmonary arteries; IL-1 and PDGF levels; or survival. Conclusions: On the basis of our results, we cannot conclude that the monocrotaline+pneumonectomy model is superior to the monocrotaline model.

Noninvasive and invasive evaluation of cardiac dysfunction in experimental diabetes in rodents

Abstract Background Because cardiomyopathy is the leading cause of death in diabetic patients, the determination of myocardial function in diabetes mellitus is essential. In the present study, we provide an integrated approach, using noninvasive echocardiography and invasive hemodynamics to assess early changes in myocardial function of diabetic rats. Methods Diabetes was induced by streptozotocin injection (STZ, 50 mg/kg). After 30 days, echocardiography (noninvasive) at rest and invasive left ventricular (LV) cannulation at rest, during and after volume overload, were performed in diabetic (D, N = 7) and control rats (C, N = 7). The Student t test was performed to compare metabolic and echocardiographic differences between groups at 30 days. ANOVA was used to compare LV invasive measurements, followed by the Student-Newman-Keuls test. Differences were considered significant at P < 0.05 for all tests. Results Diabetes impaired LV systolic function expressed by reduced fractional shortening, ejection fraction, and velocity of circumferential fiber shortening compared with that in the control group. The diabetic LV diastolic dysfunction was evidenced by diminished E-waves and increased A-waves and isovolumic relaxation time. The myocardial performance index was greater in diabetic compared with control rats, indicating impairment in diastolic and systolic function. The LV systolic pressure was reduced and the LV end-diastolic pressure was increased at rest in diabetic rats. The volume overload increased LVEDP in both groups, while LVEDP remained increased after volume overload only in diabetic rats. Conclusion These results suggest that STZ-diabetes induces systolic and diastolic dysfunction at rest, and reduces the capacity for cardiac adjustment to volume overload. In addition, it was also demonstrated that rodent echocardiography can be a useful, clinically relevant tool for the study of initial diabetic cardiomyopathy manifestations in asymptomatic patients.

B cells Can Modulate the CD8 Memory T Cell after DNA Vaccination Against Experimental Tuberculosis

Abstract Background Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown. Methods In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge. Results In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice. Conclusions These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.

Small volume of hypertonic saline as the initial fluid replacement in experimental hypodynamic sepsis

Abstract Introduction We conducted the present study to examine the effects of hypertonic saline solution (7.5%) on cardiovascular function and splanchnic perfusion in experimental sepsis. Methods Anesthetized and mechanically ventilated mongrel dogs received an intravenous infusion of live Escherichia coli over 30 minutes. After 30 minutes, they were randomized to receive lactated Ringer's solution 32 ml/kg (LR; n = 7) over 30 minutes or 7.5% hypertonic saline solution 4 ml/kg (HS; n = 8) over 5 minutes. They were observed without additional interventions for 120 minutes. Cardiac output (CO), mean arterial pressure (MAP), portal and renal blood flow (PBF and RBF, respectively), gastric partial pressure of CO2 (pCO2; gas tonometry), blood gases and lactate levels were assessed. Results E. coli infusion promoted significant reductions in CO, MAP, PBF and RBF (approximately 45%, 12%, 45% and 25%, respectively) accompanied by an increase in lactate levels and systemic and mesenteric oxygen extraction (sO2ER and mO2ER). Widening of venous-arterial (approximately 15 mmHg), portal-arterial (approximately 18 mmHg) and gastric mucosal-arterial (approximately 55 mmHg) pCO2 gradients were also observed. LR and HS infusion transiently improved systemic and regional blood flow. However, HS infusion was associated with a significant and sustained reduction of systemic (18 ± 2.6 versus 38 ± 5.9%) and mesenteric oxygen extraction (18.5 ± 1.9 versus 36.5 ± 5.4%), without worsening other perfusional markers. Conclusion A large volume of LR or a small volume of HS promoted similar transient hemodynamic benefits in this sepsis model. However, a single bolus of HS did promote sustained reduction of systemic and mesenteric oxygen extraction, suggesting that hypertonic saline solution could be used as a salutary intervention during fluid resuscitation in septic patients.

Effects of overinflation on procollagen type III expression in experimental acute lung injury

Abstract Introduction In acute lung injury (ALI), elevation of procollagen type III (PC III) occurs early and has an adverse impact on outcome. We examined whether different high-inflation strategies of mechanical ventilation (MV) in oleic acid (OA) ALI alter regional expression of PC III. Methods We designed an experimental, randomized, and controlled protocol in which rats were allocated to two control groups (no injury, recruited [alveolar recruitment maneuver after tracheotomy without MV; n = 4 rats] and control [n = 5 rats]) or four injured groups (one exposed to OA only [n = 10 rats] and three OA-injured and ventilated). The three OA-injured groups were ventilated for 1 hour according to the following strategies: LVHP-S (low volume-high positive end-expiratory pressure [PEEP], supine; n = 10 rats, tidal volume [VT] = 8 ml/kg, PEEP = 12 cm H2O), HVLP-S (high volume-low PEEP, supine; n = 10 rats, VT = 20 ml/kg, PEEP = 5 cm H2O), and HVLP-P (high volume-low PEEP, prone; n = 10 rats). Northern blot analysis for PC III and interleukin-1-beta (IL-1β) and polymorphonuclear infiltration index (PMI) counting were performed in nondependent and dependent regions. Regional differences between groups were assessed by two-way analysis of variance after logarithmic transformation and post hoc tests. Results A significant interaction for group and region effects was observed for PC III (p = 0.012) with higher expression in the nondependent region for HVLP-S and LVHP-S, intermediate for OA and HVLP-P, and lower for control (group effect, p < 0.00001, partial η2 = 0.767; region effect, p = 0.0007, partial η2 = 0.091). We found high expression of IL-1β (group effect, p < 0.00001, partial η2 = 0.944) in the OA, HVLP-S, and HVLP-P groups without regional differences (p = 0.16). PMI behaved similarly (group effect, p < 0.00001, partial η2 = 0.832). Conclusion PC III expression is higher in nondependent regions and in ventilatory strategies that caused overdistension. This response was partially attenuated by prone positioning.

Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract Introduction Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway. Methods During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay. Results Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α. Conclusion We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.

Evidence supporting a protective role for Th9 and Th22 cytokines in human and experimental periapical lesions

Introduction: The development of periapical granulomas is dependent on the host response and involves Th1, Th2, Th17, and Treg-related cytokines. The discovery of new Th9 and Th22 subsets, with important immunomodulatory roles mediated by interleukin (IL)-9 and IL-22, respectively, emphasizes the need for reevaluation of current cytokine paradigms in context of periapical lesions. We investigated the expression of IL-9 and IL-22 in active and stable human granulomas and throughout experimental lesion development in mice. Methods: Periapical granulomas (N = 83) and control specimens (N = 24) were evaluated regarding the expression of IL-9 and IL-22 via realtime polymerase chain reaction. Experimental periapical lesions were induced in mice (pulp exposure and bacterial inoculation) and the lesions evolution correlation with IL-9 and IL-22 expression kinetics was evaluated. Results: IL-9 and IL-22 mRNA expression was higher in periapical lesions than in control samples; higher levels of IL-9 and IL-22 were observed in inactive than in active lesions. In the experimental lesions model, increasing levels of IL-9 and IL-22 mRNA were detected in the lesions, and inverse correlations were found between IL-9 and IL-22 and the increase of lesion area in the different time point intervals. Conclusions: Our results suggest that Th9 and Th22 pathways may contribute to human and experimental periapical lesion stability