Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Intramyocardial injection of human adipose-derived stem cells ameliorates cognitive deficit by regulating oxidative stress-mediated hippocampal damage after myocardial infarction.

Cognitive impairment is a serious side effect of post-myocardial infarction (MI) course. We have recently demonstrated that human adipose-derived stem cells (hADSCs) ameliorated myocardial injury after MI by attenuating reactive oxygen species (ROS) levels. Here, we studied whether the beneficial effects of intramyocardial hADSC transplantation can extend to the brain and how they may attenuate cognitive dysfunction via modulating ROS after MI. After coronary ligation, male Wistar rats were randomized via an intramyocardial route to receive either vehicle, hADSC transplantation (1 × 106 cells), or the combination of hADSCs and 3-Morpholinosydnonimine (SIN-1, a peroxynitrite donor). Whether hADSCs migrated into the hippocampus was assessed by using human-specific primers in qPCR reactions. Passive avoidance test was used to assess cognitive performance. Postinfarction was associated with increased oxidative stress in the myocardium, circulation, and hippocampus. This was coupled with decreased numbers of dendritic spines as well as a significant downregulation of synaptic plasticity consisting of synaptophysin and PSD95. Step-through latency during passive avoidance test was impaired in vehicle-treated rats after MI. Intramyocardial hADSC injection exerted therapeutic benefits in improving cardiac function and cognitive impairment. None of hADSCs was detected in rat's hippocampus at the 3rd day after intramyocardial injection. The beneficial effects of hADSCs on MI-induced histological and cognitive changes were abolished after adding SIN-1. MI-induced ROS attacked the hippocampus to induce neurodegeneration, resulting in cognitive deficit. The remotely intramyocardial administration of hADSCs has the capacity of improved synaptic neuroplasticity in the hippocampus mediated by ROS, not the cell engraftment, after MI. KEY MESSAGES: Human adipose-derived stem cells (hADSCs) ameliorated injury after myocardial infarction by attenuating reactive oxygen species (ROS) levels. Intramyocardial administration of hADSCs remotely exerted therapeutic benefits in improving cognitive impairment after myocardial infarction. The improved synaptic neuroplasticity in the hippocampus was mediated by hADSC-inhibiting ROS, not by the stem cell engraftment.

Oxidative Stress in Patients before and after On-Pump and Off-Pump Coronary Artery Bypass Grafting: Relationship with Syntax Score.

OBJECTIVE: Coronary artery bypass grafting (CABG) represents the significant source of increased oxidative stress (OS). We aimed to follow the OS status parameters (i.e., ischemia-modified albumin (IMA), malondialdehyde (MDA), superoxide anion, prooxidant-antioxidant balance (PAB), total oxidant status (TOS), total antioxidant status (TAS), and superoxide-dismutase (SOD)) change through the predefined study times in two different surgical procedures, i.e., cardiopulmonary bypass (CPB) and off-pump coronary artery bypass grafting (OPCAB). Additionally, we aimed to investigate those OS status parameters in specific study times according to SYNTAX score (SS), an established angiographic score for evaluating the extensity and severity of coronary artery disease. Patients and Methods. A total of 107 patients that were planned to undergo CABG were included (i.e., 47 patients in OPCAB and 60 patients in CPB group). Blood samples were taken at 6 time intervals: before surgery (t1), immediately after intervention (t2), 6 h (t3), 24 h (t4), 48 h (t5), and 96 h after termination of the operation (t6). RESULTS: IMA levels were higher in CPB than that in OPCAB baseline and rose in CPB group in t2 point. TOS decreased in both study groups, compared to baseline values, but without statistical significance. Superoxide anion and PAB significantly increased in t3-t6 study times, in both groups. MDA significantly increased only in CPB group in t5 and t6 interval. MDA was significantly higher in CPB group compared to OPCAB in t6 study point. CPB patients had significantly lower TAS compared to OPCAB patients at the beginning and in t2 and t3 study points. They also had significantly lower SOD activities compared to OPCAB, baseline, and in several study points. Moreover, TAS, SOD, and TAS/TOS ratio were significantly lower, whereas PAB and TOS/TAS were significantly higher in patients with high SS compared to corresponding groups. SOD activity, IMA, and TAS level were the best predictors of high SS. CONCLUSION: CPB patients were in more severe ischemia baseline than OPCAB group and IMA rose in CPB patients immediately after the surgery end, but not later. Also, the antioxidant status was significantly lower, whereas the prooxidant status was significantly higher in patients with high SS compared to corresponding groups. SOD activity, IMA, and TAS level were the best predictors of CAD (as determined with SS), showing that SOD and IMA had very good discriminatory capability towards higher SS status.

Correlation between human health and reactive oxygen species produced in blood: a long-term chemiluminescence and fluorescence analysis.

The previous slide-glass type system could simultaneously detect reactive and highly reactive oxygen species, i.e., superoxide radicals (O2-·) and hypochlorite ions (OCl-) elicited from leucocytes in sample blood, but had some drawbacks, i.e., signal noise from air-flow stirring, potential biohazard risks, etc. because of open samples placed on a slide glass. We overcame these drawbacks by adopting a fluidic-chip container in a new system, which resulted in higher sensitivity and more stable measurements. Using the new system, we conducted a pilot study on nominally healthy volunteers to find whether or not the monitored activities of leukocytes can distinguish more or less unhealthy conditions from healthy ones. At first, healthy volunteers of both genders and of various ages showed that the fluctuation magnitudes (%) of O2-· and OCl- were nearly similar to each other and to that of the neutrophil count fluctuation. These parameters sometimes exceeded the healthy fluctuation range. By comparing these large fluctuations with the data of an inflammation marker C-reactive protein (CRP), the neutrophil count fluctuation and the timings/symptoms of abnormalities found in questionnaire, we could gain information suggesting the factors causing the large fluctuations. The new system could detect bodily abnormalities earlier than CRP or self-aware symptoms.

The effect of gamma irradiation on platelet redox state during storage.

BACKGROUND: As a method with insignificant adverse effects on in vitro quality of platelet concentrates (PCs), gamma irradiation is applied to abrogate the risk of transfusion-associated graft-vs-host disease in vulnerable recipients. However, there is some evidence of lower posttransfusion responses and proteomic alterations in gamma-irradiated platelets (PLTs), which raises some questions about their quality, safety, and efficacy. Since reactive oxygen species (ROS) are considered as markers of PLT storage lesion (PSL), the study presented here investigated oxidant state in gamma-irradiated PCs. STUDY DESIGN AND METHODS: PLT-rich plasma PC was split into two bags, one kept as control while other was subjected to gamma irradiation. Within 7 days of storage, the levels of intra-PLT superoxide, H2 O2 , mitochondrial ROS, P-selectin expression, and phosphatidylserine (PS) exposure were detected by flow cytometry while intracellular reduced glutathione (GSH), glucose concentration, and lactate dehydrogenase (LDH) activity were measured by enzymocolorimetric method. RESULTS: GSH decreased, while ROS generation and LDH activity increased, during storage. Gamma irradiation significantly attenuated GSH whereas increased ROS generation in earlier and later stages of storage associated with either P-selectin or PS exposure increments. CONCLUSION: Gamma irradiation can significantly increase cytosolic ROS generation in two distinct phases, one upon irradiation and another later in longer-stored PCs. While earlier ROS influx seems to be governed by direct effect of irradiation, the second phase of oxidant stress is presumably due to the storage-dependent PLT activation. Intriguingly, these observations were also in line with early P-selectin increments and increased PS exposure in longer-stored PLTs. Given the mutual link between ROS generation and PLT activation, further investigation is required to explore the effect of gamma irradiation on the induction of PSL.

NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis.

OBJECTIVE: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. CONCLUSIONS: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

In Vitro Interaction of 5-Aminoorotic Acid and Its Gallium(III) Complex with Superoxide Radical, Generated by Two Model Systems.

Increased levels of the superoxide radical are associated with oxidative damage to healthy tissues and with elimination of malignant cells in a living body. It is desirable that a chemotherapeutic combines pro-oxidant behavior around and inside tumors with antioxidant action near healthy cells. A complex consisting of a pro-oxidant cation and antioxidant ligands could be a potential anticancer agent. Ga(III) salts are known anticancer substances, and 5-aminoorotic acid (HAOA) is a ligand with antioxidant properties. The in vitro effects of HAOA and its complex with Ga(III) (gallium(III) 5-aminoorotate (GaAOA)) on the in vitro accumulation of superoxide and other free radicals were estimated. Model systems such as potassium superoxide (KO2), xanthine/xanthine oxidase (X/XO), and rat blood serum were utilized. Data suggested better antioxidant effect of GaAOA compared to HAOA. Evidently, all three ligands of GaAOA participated in the scavenging of superoxide. The effects in rat blood serum were more nuanced, considering the chemical and biochemical complexity of this model system. It was observed that the free-radical-scavenging action of both compounds investigated may be manifested via both hydrogen donation and electron transfer pathways. It was proposed that the radical-scavenging activities (RSAs) of HAOA and its complex with Ga(III) may be due to a complex process, depending on the concentration, and on the environment, nature, and size of the free radical. The electron transfer pathway was considered as more probable in comparison to hydrogen donation in the scavenging of superoxide by 5-aminoorotic acid and its gallium(III) complex.

Effect of heat stress on superoxide anion production in native and crossbred cattle under in vitro whole blood culture model.

Impact of global warming on the dairy industry has gained attention due to huge economic losses through low production and fertility caused by heat stress. Exposure to hyperthermia provokes a series of complex responses in mammals which are been related to morphological and physiological alterations including the production of reactive oxygen species (ROS). A quantitative spectrophotometric based nitroblue tetrazolium (NBT) reduction assay was used to estimate the superoxide anion (•O2-) level in heat stressed (at 42 °C) whole blood cultures of native and crossbred bulls (Sahiwal and Frieswal), in vitro. The breed effect in the kinetics of •O2- production at different time periods of continual heat stress was analyzed by repeated measures ANOVA. Comparison between different time periods in reference to 37 °C was analyzed by paired t-test. The •O2- level was significantly different (p < 0.05) between cells at 37 °C and 42 °C at different periods of incubation. Kinetics study showed increment of •O2- production on the acute phase of stress followed by a reduction in both Sahiwal and Frieswal breeds. In Sahiwal breed, the inflated superoxide level continued abated till 4 h and raised again at 6 h, while in Frieswal •O2- level reverted to raise sooner with in 2 h of incubation itself. Contrarily, kinetic of •O2- level in plasma showed a significant reduction (p < 0.001) at 30 min of 42 °C incubation followed by increment of •O2- level. Further, the breed variation was significant (p < 0.05) and a significant high reduction of •O2- level was observed in Sahiwal breed. Our finding indicates that, a better and longer •O2- production homeostasis and higher plasma scavenging ability of native breed may be one of the reasons for the higher thermal tolerance of these breeds in tropical climate.

A Renal-Clearable Duplex Optical Reporter for Real-Time Imaging of Contrast-Induced Acute Kidney Injury.

Despite its high morbidity and mortality, contrast-induced acute kidney injury (CIAKI) remains a diagnostic dilemma because it relies on in vitro detection of insensitive late-stage blood and urinary biomarkers. We report the synthesis of an activatable duplex reporter (ADR) for real-time in vivo imaging of CIAKI. ADR is equipped with chemiluminescence and near-infrared fluorescence (NIRF) signaling channels that can be activated by oxidative stress (superoxide anion, O2.- ) and lysosomal damage (N-acetyl-ß-d-glucosaminidase, NAG), respectively. By virtue of its high renal clearance efficiency (80 % injected doses after 24 h injection), ADR detects sequential upregulation of O2.- and NAG in the kidneys of living mice prior to a significant decrease in glomerular filtration rate (GFR) and tissue damage in the course of CIAKI. ADR outperforms the typical clinical assays and detects CIAKI at least 8 h (NIRF) and up to 16 h (chemiluminescence) earlier.

Prognostic Value of Oxidative Stress Markers in Patients with Pulmonary Arterial or Chronic Thromboembolic Pulmonary Hypertension.

Oxidative stress is regarded to play a crucial role in the pathophysiology of pulmonary arterial hypertension (PAH) and inoperable chronic thromboembolic pulmonary hypertension (CTEPH). This study evaluated the prognostic value of serum oxidative stress markers (malondialdehyde (MDA), total antioxidant capacity (TAC), catalase activity (CAT), and superoxide activity (SOD)) in patients with PAH and CTEPH (n = 45). During 13 months of follow-up (median 9 months), clinical deterioration occurred in 14 patients (including 2 deaths). On the Cox regression analysis, MDA, TAC, and CAT were associated with clinical deterioration (p = 0.0068, HR = 1.42, 95% CI: 1.10-1.82; p = 0.0038, HR = 0.033, 95% CI: 0.0032-0.33; and p = 0.046, HR = 0.20, 95% CI: 0.04-0.98, respectively). There was no significant difference in SOD (p = 0.53, HR = 0.97, 95% CI: 0.87-1.08). The cut-off value derived from ROC curve analysis was 3.79 µM (p = 0.0048, AUC = 0.76, 95% CI: 0.62-0.91) for MDA, 0.49 mM (p = 0.027, AUC = 0.71, 95% CI: 0.18-0.47) for TAC, and 1.34 U/L (p = 0.029, AUC = 0.71, 95% CI: 0.55-0.86) for CAT. MDA in the group with deterioration was higher (p = 0.0041), while TAC as well as CAT were lower (p = 0.027 and p = 0.028, respectively) when compared to stable patients. Survival without clinical deterioration was significantly longer in patients with lower MDA (p = 0.037, HR = 0.37, 95% CI: 0.12-1.14, log-rank), higher TAC (p = 0.0018, HR = 0.19, 95% CI: 0.06-0.60, log-rank), and higher CAT (p = 0.044, HR = 0.31 95% CI: 0.11-0.88, log-rank). Markers of oxidative stress such as MDA, TAC, and CAT were associated with adverse clinical outcomes in patients with PAH and inoperable or residual CTEPH.

Angiotensin-(1-7) attenuates organ injury and mortality in rats with polymicrobial sepsis.

BACKGROUND: Sepsis and related multiple organ dysfunction result in high morbidity and mortality. Angiotensin (Ang)-(1-7), a biologically active peptide, has various opposing effects of Ang II. Because the effect of Ang-(1-7) on sepsis is unknown, in this study we aimed to determine the impact of Ang-(1-7) on pathophysiologic changes in a clinically relevant model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). METHODS: Sepsis was induced by CLP in rats under anesthesia. Rats were randomized to one of the following five groups: (1) sham-operated group, (2) Ang-(1-7) (1 mg/kg intravenously infused for 1 h) at 3 h and 6 h after sham operation, (3) CLP, (4) Ang-(1-7) at 3 h after CLP, and (5) Ang-(1-7) at 3 h and 6 h after CLP. Rats were observed for 24 h after CLP surgery and then killed for subsequent histological examination. RESULTS: Ang-(1-7) significantly improved the survival of septic rats (83.3% vs. 36.4% at 24 h following CLP; p = 0.009). Ang-(1-7) attenuated the CLP-induced decreased arterial pressure and organ dysfunction, indicated by diminished biochemical variables and fewer histological changes. Ang-(1-7) significantly reduced the level of plasma interleukin-6 and pulmonary superoxide production (p < 0.05). Moreover, caspase-3 and cytoplasmic IκB expression in liver was significantly lower in the Ang-(1-7)-treated CLP rats (p < 0.05). CONCLUSIONS: In this clinically relevant model of sepsis, Ang-(1-7) ameliorates CLP-induced organ dysfunction and improves survival, possibly through suppressing the inflammatory response, oxidative stress, and apoptosis, suggesting that Ang-(1-7) could be a potential novel therapeutic approach to treatment of peritonitis and polymicrobial sepsis.

Oxidative stress in healthy pregnancy and preeclampsia is linked to chronic inflammation, iron status and vascular function.

BACKGROUND: During normal pregnancy, placental oxidative stress (OS) is present during all three trimesters and is necessary to obtain normal cell function. However, if OS reaches a certain level, pregnancy complications might arise. In preeclampsia (PE), a dangerous pregnancy specific hypertensive disorder, OS induced in the ischemic placenta causes a systemic inflammatory response and activates maternal endothelial cells. In this study, we aimed to quantify superoxide concentrations (as a measure of systemic OS) using electron paramagnetic resonance (EPR) and correlate them to markers of systemic inflammation, iron status and vascular function. METHODS: Fifty-nine women with a healthy pregnancy (HP), 10 non-pregnant controls (NP) and 28 PE patients (32±3.3weeks) were included. During HP, blood samples for superoxide, neutrophil to lymphocyte ratio (NLR), mean platelet volume (MPV) and iron status were taken at 10, 25 and 39 weeks. Vascular measurements for arterial stiffness (carotid-femoral pulse wave velocity (CF-PWV), augmentation index (AIx), augmentation Pressure (AP)) and microvascular endothelial function (reactive hyperemia index (RHI)) were performed at 35 weeks. In PE, all measurements were performed at diagnosis. CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) was used as spin probe for EPR, since the formed CM radical corresponds to the amount of superoxide. RESULTS: Superoxide concentration remains stable during pregnancy (p = 0.92), but is significantly higher compared to the NP controls (p<0.0001). At 25 weeks, there is a significant positive correlation between superoxide and ferritin concentration. (p = 0.04) In PE, superoxide, systemic inflammation and iron status are much higher compared to HP (all p<0.001). During HP, superoxide concentrations correlate significantly with arterial stiffness (all p<0.04), while in PE superoxide is significantly correlated to microvascular endothelial function (p = 0.03). CONCLUSIONS: During HP there is an increased but stable oxidative environment, which is correlated to ferritin concentration. If superoxide levels increase, there is an augmentation in arterial stiffness. In PE pregnancies, systemic inflammation and superoxide concentrations are higher and result in a deterioration of endothelial function. Together, these findings support the hypothesis that vascular function is directly linked to the amount of OS and that measurement of OS in combination with vascular function tests might be used in the prediction of PE.

Oxidative stress and endothelial function in normal pregnancy versus pre-eclampsia, a combined longitudinal and case control study.

BACKGROUND: Pre-eclampsia (PE) is related to an impaired endothelial function. Endothelial dysfunction accounts for altered vascular reactivity, activation of the coagulation cascade and loss of vascular integrity. Impaired endothelial function originates from production of inflammatory and cytotoxic factors by the ischemic placenta and results in systemic oxidative stress (OS) and an altered bioavailability of nitric oxide (•NO). The free radical •NO, is an endogenous endothelium-derived relaxing factor influencing endothelial function. In placental circulation, endothelial release of •NO dilates the fetal placental vascular bed, ensuring feto-maternal exchange. The Endopreg study was designed to evaluate in vivo endothelial function and to quantify in vitro OS in normal and pre-eclamptic pregnancies. METHODS/DESIGN: The study is divided into two arms, a prospective longitudinal study and a matched case control study. In the longitudinal study, pregnant patients ≥18 years old with a singleton pregnancy will be followed throughout pregnancy and until 6 months post-partum. In the case control study, cases with PE will be compared to matched normotensive pregnant women. Maternal blood concentration of superoxide (O2•) and placental concentration of •NO will be determined using EPR (electron paramagnetic resonance). Endothelial function and arterial stiffness will be evaluated using respectively Peripheral Arterial Tonometry (PAT), Flow-Mediated Dilatation (FMD) and applanation tonometry. Placental expression of eNOS (endothelial NOS) will be determined using immune-histochemical staining. Target recruitment will be 110 patients for the longitudinal study and 90 patients in the case-control study. DISCUSSION: The results of Endopreg will provide longitudinal information on in vivo endothelial function and in vitro OS during normal pregnancy and PE. Adoption of these vascular tests in clinical practice potentially predicts patients at risk to develop cardiovascular events later in life after PE pregnancies. •NO, O2•- and eNOS measurements provide further inside in the pathophysiology of PE. TRIAL REGISTRATION: This trial has been registered on clinicaltrials.gov. ClinicalTrials.gov Identifier: NCT02603913 . Registered October 2015.

Effects of atorvastatin and simvastatin on oxidative stress in diet-induced hyperhomocysteinemia in Wistar albino rats: a comparative study.

Considering the well-known antioxidant properties of statins, it seems important to assess their impact on major markers of oxidative stress (superoxide anion radical, nitric oxide, and index of lipid peroxidation) to compare the antioxidative potentials of atorvastatin and simvastatin during the different degrees of hyperhomocysteinemia (HHcy) in rats. This study was conducted on adult male Wistar albino rats (n = 90; 4 weeks old; 100 ± 15 g body mass) in which HHcy was achieved by dietary manipulation. For 4 weeks, the animals were fed with one of the following diets: standard rodent chow, diet enriched in methionine with no deficiency in B vitamins (folic acid, B6, and B12), or diet enriched in methionine and deficient in B vitamins (folic acid, B6, and B12). At the same time, animals were treated with atorvastatin at doses of 3 mg/kg/day i.p. or simvastatin at doses of 5 mg/kg/day i.p. Levels of superoxide anion radical and TBARS were significantly decreased by administration of simvastatin in normal and high-homocysteine (Hcy) groups (p < 0.05). At 4 weeks after feeding with purified diets, the concentrations of the GSH, CAT, and SOD antioxidants were significantly affected among all groups (p < 0.05). Our results indicated that statin therapy had variable effects on the redox status in hyperhomocysteinemic rats, and simvastatin demonstrated stronger antioxidant effects than did atorvastatin.

Estradiol decreases blood pressure in association with redox regulation in preeclampsia.

In this study, we tested a hypothesis that a short-term estradiol therapy may reduce blood pressure in preeclampsia by modulating plasma oxidative stress. The intramuscular injections of 10 mg 17-beta-estradiol were prescribed to preeclamptic pregnant women during the 3-day therapy before a labor induction. The analyses of mean arterial pressure (MAP), serum estradiol concentrations, plasma superoxide anion (O2.), hydrogen peroxide (H2O2), nitrites (NO2-), and peroxynitrite (ONOO-) were conducted before and during the therapy. We found that the plasma concentrations of oxidative stress markers, such as O2- and H2O2, are higher in preeclampsia and positively correlated with the MAP value. Moreover, it was shown that the plasma concentration of NO2- as an indicator of NO levels is higher in preeclampsia. A short-term intramuscular application of estradiol decreases the MAP value and the plasma concentration of O.-, H2O2, NO2-, and ONOO- in preeclampsia. A positive correlation between the decrease of MAP values and the decrease of plasma concentrations of O2-, H2O2, and ONOO- was found in preeclampsia during a short-term estradiol therapy. We conclude that the short-term estradiol therapy decreases the MAP value in preeclampsia by modulating the plasma oxidative stress. We speculate that the estradiol metabolism in preeclampsia is an important mechanism that contributes to vascular dysfunction.

PROTECTIVE EFFECT OF MORINGA PEREGRINA LEAVES EXTRACT ON ACETAMINOPHEN -INDUCED LIVER TOXICITY IN ALBINO RATS.

BACKGROUND: Acetaminophen is a common antipyretic drug but at overdose can cause severe hepatotoxicity that may further develop into liver failure and hepatic centrilobular necrosis in experimental animals and humans. This study was undertaken to assess the ameliorative role of Moringa peregrina leaves extract against acetaminophen toxicity in rats. MATERIALS AND METHODS: Induction of hepatotoxicity was done by chronic oral administration of acetaminophen (750 mg/kg bwt) for 4 weeks. To study the possible hepatoprotective effect, Moringa peregrina leaves extract (200 mg/kg bwt) or Silymarin (50 mg/kg bwt) was administered orally, for 4 weeks, along with acetaminophen. RESULTS: acetaminophen significantly increased serum liver enzymes and caused oxidative stress, evidenced by significantly increased tissue malondialdehyde, glutathione peroxidase, hepatic DNA fragmentation, and significant decrease of glutathione and antioxidant enzymes in liver, blood and brain. On the other hand, administration of Moringa peregrina leaves extract reversed acetaminophen-related toxic effects through: powerful malondialdehyde suppression, glutathione peroxidase normalization and stimulation of the cellular antioxidants synthesis represented by significant increase of glutathione, catalase and superoxide dismutase in liver, blood and brain, besides, DNA fragmentation was significantly decreased in the liver tissue. CONCLUSION: acetaminophen induced oxidative damage can be improved by Moringa peregrina leaves extract-treatment, due to its antioxidant potential.

Evaluation of perlite, wood shavings and corncobs for bedding material in rats.

Bedding material, which is a significant part of rodent housing, affects the health and well-being of laboratory animals. The aim of this study was to evaluate perlite as a bedding material for rodents and to compare it with wood shavings, expanded perlite and corncobs. The animals used in this experiment were 48 male and 48 female Sprague-Dawley rats. The bedding materials collected from experimental groups were analysed microbiologically. Blood samples from rats were subjected to biochemical analysis for catalase, glutathione, glutathione peroxidase, malondialdehyde, superoxide and dismutase, and foot pad skins of rats were subjected to histopathological examination. Body weight was determined at the end of the 30-day period. Perlite as the only bedding material had no effect on body weight, and it resulted in less microbial activity compared with the wood shavings, expanded perlite and corncobs. However, using perlite alone had negative effects on the skin, the moisture percentage of bedding and stress parameters. A wood shavingsperlite combination gave better results than perlite alone and appropriate perlite and other bedding material mixtures may result in bedding materials conducive to animal health and welfare. The frequency of changing the bedding material should be limited to once weekly.

Interleukin-6 as possible early marker of stress response after femoral fracture.

Bone fracture healing is a complex process which at best results in full recovery of function and structure of injured bone tissue, but all the mechanisms involved in this process, and their mutual interaction, are not fully understood. Despite advancement of surgical procedures, this type of fractures is still a major public health concern. In the last few decades, a lot of attention is focused on the oxygen-free radicals and inflammatory response markers as important factors of skeletal injury. Thus, the aim of the present study was to follow the changes in redox balance and inflammatory response in elderly patients with femoral fractures during the earliest stages of fracture healing, by measuring the values of the observed markers immediately after fracture, as well as the first, third, and seventh postoperative day. Present study was performed on a group of 65 elderly patients with femoral neck fractures, recruited from the Orthopedic Clinic, Clinical Centre Kragujevac in the period from February to May 2015. Redox status was measured spectrophotometrically and evaluated by measuring the levels of index of lipid peroxidation (measured as TBARS), nitrite (NO2-), superoxide anion radical (O2-), and hydrogen peroxide (H2O2) in plasma, while activities of corresponding antioxidative enzymes, catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) were measured in erythrocytes. The cytokine concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were determined in plasma, using ELISA assays specific for human cytokines. Our study showed that redox status and TNF-α in elderly patients with femoral fractures did not show statistically significant changes during the early phase of fracture healing. On the other hand, IL-6 increased statistically in first day after intervention. This preliminary study has shown our observations, and we hope that these results may help in better understanding mechanisms which are included at fracture healing. More importantly, this study attempted to create a platform for further research.

Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method.

BACKGROUND: Nitric oxide (NO) and its related reactive nitrogen species (RNS) and reactive oxygen species (ROS) are crucial in monocyte responses against pathogens and also in inflammatory conditions. Central to both processes is the generation of the strong oxidant peroxynitrite (ONOO) by a fast reaction between NO and superoxide anion. ONOO is a biochemical junction for ROS- and RNS cytotoxicity and causes protein nitrosylation. Circulating by-products of protein nitrosylation are early biomarkers of inflammation-based conditions, including minimal hepatic encephalopathy in cirrhotic patients (Montoliu et al., Am J Gastroenterol 2011; 106:1629-1637). In this context, we have designed a novel no-wash, no-lyse real-time flow cytometry assay to detect and follow-up the NO- and superoxide-driven generation of ONOO in peripheral blood monocytes. METHODS: Whole blood samples were stained with CD45 and CD14 antibodies plus one of a series of fluorescent probes sensitive to RNS, ROS, or glutathione, namely 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, dihydrorhodamine 123, MitoSOX Red, dihydroethidium, and 5-chloromethylfluorescein diacetate. Samples were exposed sequentially to a NO donor and three different superoxide donors, and analyzed in real time by kinetic flow cytometry. Relevant kinetic descriptors, such as the rate of fluorescence change, were calculated from the kinetic plot. RESULTS: The generation of ONOO, which consumes both NO and superoxide, led to a decrease in the intensity of the cellular fluorescence of the probes sensitive to these molecules. CONCLUSION: This is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. © 2015 International Clinical Cytometry Society.

Inflammatory Events and Oxidant Production in the Diaphragm, Gastrocnemius, and Blood of Rats Exposed to Chronic Intermittent Hypoxia: Therapeutic Strategies.

We hypothesized that inflammatory events and reactive oxygen species (ROS) production may be differentially expressed in respiratory and limb muscles, and blood of a chronic intermittent hypoxia (CIH) experimental model and that antioxidants and TNF-alpha blockade may influence those events. In blood, diaphragm, and gastrocnemius of rats non-invasively exposed to CIH (10% hypoxia, 2 h/day, 14 consecutive days) with/without concomitant treatment with either anti-TNF-alpha antibody (infliximab) or N-acetyl cysteine (NAC), inflammatory cytokines, superoxide anion production, muscle structural abnormalities, and fiber-type composition were assessed. Compared to non-exposed controls, in CIH-exposed rats, body weight gain was reduced, TNF-alpha, IL-1beta, IL-6, and interferon-gamma levels were increased in diaphragm, TNF-alpha, and IL-1 beta plasma levels were greater, systemic and muscle superoxide anion production was higher, diaphragm and gastrocnemius inflammatory cells and internal nuclei were higher, and muscle fiber-type and morphometry remained unmodified. CIH rats treated with infliximab further increased TNF-alpha, IL-1beta, IL-6, and interferon-gamma diaphragm levels, whereas NAC induced a reduction only in TNF-alpha and IL-1beta levels in diaphragm and plasma. Infliximab and NAC elicited a significant decline in superoxide anion production in diaphragm, gastrocnemius, and plasma, while inducing a further increase in inflammatory cells and internal nuclei in both muscles. Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH-exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases. J. Cell. Physiol. 232: 1165-1175, 2017. © 2016 Wiley Periodicals, Inc.