Sophia Lavrentiadou

Effect of long-term dietary administration of oregano on the alleviation of carbon tetrachloride-induced oxidative stress in rats.

This study aimed at evaluating the protective effect of long-term dietary oregano on the alleviation of carbon tetrachloride-induced oxidative stress in rats. Twenty-four female Wistar rats were allocated to four groups of six animals each. Groups 1 (control) and 2 (CCl 4) were fed a basal diet, while groups 3 (oregano) and 4 (oregano + CCl 4) were fed the basal diet supplemented further with ground oregano at 1% level. Following six-week feeding, the rats of groups 2 and 4 were given a single intraperitoneal injection of CCl 4 at a dose of 1 mL/kg bw. Six hours after the CCl 4 injection, all animals were sacrificed, and serum, liver, kidney, and heart tissue samples were collected. Analysis results showed that the addition of oregano significantly increased the total phenolic content and the Trolox equivalent antioxidant capacity of the basal diet but had no effect on its lipid peroxidation index. Treatment with CCl 4 of rats from the CCl 4 group caused a significant increase in aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) in serum, whereas it decreased cholesterol and triglyceride content as compared to the control. It also increased the lipid peroxidation index and decreased the scavenging activities of the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS) radical cation, the hydroxyl anion radical, the superoxide anion radical, and the hydrogen peroxide in all tested tissues, as compared to that of the control. Without CCl 4 treatment, diet supplementation with oregano had no effect on these biochemical parameters, excluding the hydroxyl radical scavenging activity, which was increased in all tested tissues as compared to that of the control. Feeding oregano before CCl 4 treatment resulted in a significant decline of the increase in AST, ALT, and ALP activities ( P < 0.05 vs CCl 4 group), but the recorded values could not attain those of the control group ( P < 0.05 vs control group). It significantly increased the reduced cholesterol and triglycerides ( P < 0.05 vs CCl 4 group) to values not differing from those of the control. It also resulted in a significant reduction of the increased malondialdehyde ( P < 0.05 vs CCl 4 group) to values that could not attain the levels of the control but had no significant effect ( P > 0.05) on the reduced ABTS radical cation scavenging activity. It increased significantly the reduced hydroxyl anion radical scavenging activity ( P < 0.05 vs CCl 4 group) to values that could not attain those of the control in all tested tissues except kidney. Additionally, it resulted in a significant elevation of the decreased superoxide anion radical scavenging activity in serum and liver but had no effect in kidney and heart, whereas it also resulted in a significant elevation of the decreased hydrogen peroxide scavenging activity in liver, kidney, and heart but had no effect in serum. These results suggest that dietary oregano may effectively improve the impaired antioxidant status in CCl 4-induced toxicity in rats.

Urokinase-Type Plasminogen Activator Deficiency Promotes Neoplasmatogenesis in the Colon of Mice

Urokinase-type plasminogen activator (uPA) participates in cancer-related biologic processes, such as wound healing and inflammation. The present study aimed to investigate the effect of uPA deficiency on the long-term outcome of early life episodes of dextran sodium sulfate (DSS)–induced colitis in mice. Wild-type (WT) and uPA-deficient (uPA−/−) BALB/c mice were treated with DSS or remained untreated. Mice were necropsied either 1 week or 7 months after DSS treatment. Colon samples were analyzed by histopathology, immunohistochemistry, ELISA, and real-time polymerase chain reaction. At 7 months, with no colitis evident, half of the uPA−/− mice had large colonic polypoid adenomas, whereas WT mice did not. One week after DSS treatment, there were typical DSS-induced colitis lesions in both WT and uPA−/− mice. The affected colon of uPA−/− mice, however, had features of delayed ulcer re-epithelialization and dysplastic lesions of higher grade developing on the basis of a significantly altered mucosal inflammatory milieu. The later was characterized by more neutrophils and macrophages, less regulatory T cells (Treg), significantly upregulated cytokines, including interleukin-6 (IL-6), IL-17, tumor necrosis factor-α, and IL-10, and lower levels of active transforming growth factor–β1 (TGF-β1) compared to WT mice. Dysfunctional Treg, more robust protumorigenic inflammatory events, and an inherited inability to produce adequate amounts of extracellular active TGF-β1 due to uPA deficiency are interlinked as probable explanations for the inflammatory-induced neoplasmatogenesis in the colon of uPA−/− mice.

CCl4 induces tissue-type plasminogen activator in rat brain; protective effects of oregano, rosemary or vitamin E

The high metabolic rate and relatively low antioxidant defenses of the lipid-rich brain tissue render it highly susceptible to reactive oxygen species (ROS) and oxidative stress, whereas the implication of ROS in the pathogenesis of several diseases in the central nervous system is well-established. The plasminogen activator (PA) system is a key modulator of extracellular proteolysis, extracellular matrix remodeling and neuronal cell signaling and has been implicated in the pathogenesis of these diseases. This study evaluates the role of tissue-type PA (t-PA) in oxidative stress and the protective role of dietary antioxidants in the rat brain. We used the CCl4 experimental model of ROS-induced lipid peroxidation and evaluated the antioxidant effect of oregano, rosemary or vitamin E. CCl4-treated Wistar rats exhibited elevated brain t-PA activity, which was decreased upon long-term administration of oregano, rosemary or vitamin E. PA inhibitor-1 (PAI-1) activity was also slightly elevated by CCl4, but this increase was not affected by the antioxidants. We hypothesize that the CCl4-induced t-PA activity indicates extracellular proteolytic activity that may be linked to neuronal cell death and brain damage. Vitamin E or antioxidants present in oregano or rosemary are effective in inhibiting t-PA elevation and can be considered as a potential protection against neuronal damage.

In vivo image analysis of BoHV-4-based vector in mice.

Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver.

Crocetin administration ameliorates endotoxin-induced disseminated intravascular coagulation in rabbits.

Disseminated intravascular coagulation (DIC), a life-threatening secondary complication in several diseases, is characterized by large amounts of thrombin that lead to fibrin deposition and microthrombus formation throughout the microcirculation. Recent in-vitro studies suggest that crocin, crocetin or safranal, carotenoid constituents of the spice Crocus sativus L. (saffron), have antithrombotic properties, especially anti-Xa activity. The present study was designed to evaluate the effect of crocetin on thrombosis procedure using a rabbit model of bacterial endotoxin-induced DIC. Crocetin administration (3 mg/kg), 30 min before the beginning of endotoxin infusion, improved DIC-related haemostatic indices such as platelet blood counts (P ⩽ 0.05), blood plasma fibrinogen and protein C concentration (P ⩽ 0.05). In addition, it ameliorated DIC-associated disease and fibrin deposition in the glomeruli (P ⩽ 0.05). These results indicate that crocetin reveals a preventive antithrombotic role in vivo and prescribe further investigation on the possibility of developing crocetin-based DIC treatment modalities.

Seasonal Variation of Plasminogen Activator Activity in Spermatozoa and Seminal Plasma of Boar, Buck, Bull and Stallion

Plasminogen activators (PA) are proteolytic enzymes present in the spermatozoa and seminal plasma of various species. They play a role in the binding of the spermatozoon and its penetration through the layers surrounding the oocyte. Plasminogen activator activity (PAA) is modulated by hormones that have a seasonal variation, such as testosterone and melatonin. The present study investigates the seasonal variation of PA activity in sperm extracts and seminal plasma of four farm animal species: boar, buck, bull and stallion. Semen samples were collected every second week during a 12-month period and PAA was determined. With respect to sperm enzyme activity, the boar showed a peak from late January until the beginning of April, whereas the activity in the bull was at the highest levels from April until October and gradually declined during autumn and winter period. Plasminogen activator activity of stallion spermatozoa peaked during March and April, and remained low throughout the rest of the year, whereas in the buck sperm, PAA increased from late October until the end of January. No biologically significant variation was detected regarding the seminal PAA activity in any of the species studied. While seasonality of reproduction is typically studied from the female perspective, the present data provide compelling information about a factor that may affect the reproductive ability of the male.

Enlightened Mannhemia haemolytica lung inflammation in bovinized mice

Polymorphonuclear cells diapedesis has an important contribution to the induced Mannhemia haemolytica (M. haemolytica) infection lung inflammation and IL-8 is the primary polymorphonuclear chemoattractant. Using a bovine IL-8/luciferase transiently transgenized mouse model, the orchestration among M. haemolytica, IL-8 promoter activation and neutrophilia was followed in real time by in vivo image analysis.

Protective effect of crocetin on bovine spermatozoa against oxidative stress during in vitro fertilization.

Oxidative stress is one of the major factors that contribute to poor semen quality and low rates of in vitro fertilization. Crocetin, a main constituent of saffron (Crocus sativus L.) possesses potent antioxidant activity, by scavenging reactive oxygen species (ROS) and/or enhancing the activity of intracellular antioxidant enzymes. The aim of this study was to investigate, for the first time, the effect of crocetin on the quality characteristics of bull spermatozoa and fertilization rate. For this reason, frozen/thawed bovine spermatozoa were incubated with crocetin (1, 2.5, and 5 μm), for 120 or 240 min, in the presence of a negative control, and evaluated in terms of motility, viability, acrosomal status, DNA fragmentation index, intracellular ROS, and lipid peroxidation. In order to evaluate the impact of crocetin on cleavage and blastocyst rate, the compound was added in the IVF medium at the previously identified optimal concentration (2.5 μm). The results indicate that incubation of spermatozoa with 2.5 μm of crocetin resulted in a statistically significant lower production of superoxide anion and hydrogen peroxide, lower lipid peroxidation, and in better maintenance of motility parameters, viability, and acrosomal integrity, with a very small number of cells with DNA fragmentation, compared to the other groups (p < 0.05). The presence of crocetin (2.5 μm) in the fertilization medium also resulted in a significant increase in acrosome‐reacted spermatozoa and blastocyst production, compared to the control group (p < 0.01). These data indicate that crocetin (2.5 μm) positively affects bovine sperm quality characteristics during a 240‐min incubation and improves its fertilizing ability, directly and/or indirectly, by regulating ROS concentration and lipid peroxidation.

Dietary omega-3 polyunsaturated fatty acids induce plasminogen activator activity and DNA damage in rabbit spermatozoa.

The aim of this study was to determine the effect(s) of dietary omega‐3 polyunsaturated fatty acids (ω‐3 PUFA) on rabbit semen. Adult rabbit bucks were assigned to two groups that were given two diets, a standard diet (control) and a diet supplemented with ω‐3 PUFA. Sperm samples were collected from all bucks with the use of an artificial vagina in 20‐day intervals, for a total period of 120 days. The enrichment of membranes in ω‐3 PUFA was manifested by the elevation of the 22:5 ω‐3 (docosapentaenoic acid [DPA]) levels within 40 days. This increase in DPA content did not affect semen characteristics (i.e., concentration, motility and viability). However, it was associated with the induction of lipid peroxidation in spermatozoa, as determined on the basis of the malondialdehyde content. Lipid peroxidation was associated with DNA fragmentation in ω‐3 PUFA‐enriched spermatozoa and a concomitant increase in plasminogen activator (PA) activity. The effects of ω‐3 PUFA on sperm cells were evident within 40 days of ω‐3 PUFA dietary intake and exhibited peack values on day 120. Our findings suggest that an ω‐3 PUFA‐rich diet may not affect semen characteristics; however, it may have a negative impact on the oxidative status and DNA integrity of the spermatozoa, which was associated with an induction of PAs activity.

46 PLASMINOGEN ACTIVATOR ACTIVITY IN BUFFALO IN VITRO MATURED OOCYTES AFTER VITRIFICATION-WARMING

Plasminogen activators (PA) are proteolytic enzymes that convert plasminogen into plasmin. Plasmin is involved in physiological processes such as ovulation (Liu 2004 Front. Biosci. 9, 3356–3373), cumulus cell layer expansion (Liu et al. 1986 Endocrinology 119, 1578–1587), oocyte maturation (Dow et al. 2002 Biol. Reprod. 66, 1413–1421) and fertilization (Huarte et al. 1993 Dev. Biol. 157, 539–546). Although the interest is increasing, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of the present study was to evaluate whether exposure to cryoprotectants and the vitrification procedure affect plasminogen activator activity (PAA) in buffalo in vitro-matured oocytes. A total number of 300 cumulus-oocyte complexes over 5 replicates were selected and in vitro-matured. Cumulus-oocyte complexes were mechanically stripped of their cumulus cells by gentle pipetting, washed and divided into 3 groups (20 oocytes/group, over 5 replicates). The control group consisted of fresh in vitro-matured oocytes. In the vitrification group, denuded oocytes were first exposed to 10% ethylene glycol (EG) + 10% dimethyl sulfoxide (DMSO) for 3 min, then to 20% EG + 20% DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 s. Subsequently, oocytes were warmed in a 1.25 M sucrose solution for 1 min and then in decreasing concentrations of sucrose (0.625 M, 0.42 M and 0.31 M) for 30 s each. In order to test cryoprotectant effects, oocytes were simply exposed to the vitrification and warming solutions (toxicity group). Surviving oocytes were extracted by a fine needle, centrifuged at 4000 rpm for 10 min and the supernatant was mixed with the reaction solution: TRIS-HCl 0.1 M, homologous plasminogen, the chromogenic substrate for plasmin S-2251 and incubated at 37°C for 30 min. The PAA levels were measured by a spectrophotometer (405 nm) expressed as Abs/20 oocytes. The data were analysed by the Kruskal-Wallis nonparametric test. Low levels of PAA were detected in the denuded oocytes of the control, toxicity and vitrification groups. No significant differences in mean PAA values were observed among the 3 experimental groups (0.017 ± 0.001, 0.018 ± 0.002 and 0.017 ± 0.001 units, in the control, toxicity and vitrification groups, respectively). In conclusion, cryoprotectants and the vitrification procedure do not affect the proteolytic activity linked to plasmin in in vitro-matured buffalo oocytes. The results show that the vitrification/warming procedure does not exert an effect on in vitro-matured buffalo oocytes in terms of PAA generation, a parameter that plays an important role in fertilization and in vitro embryo development. Further studies are needed to identify factors affecting the efficiency of oocyte cryopreservation.