Plant Extract of Limonium gmelinii Attenuates Oxidative Responses in Neurons, Astrocytes, and Cerebral Endothelial Cells In Vitro and Improves Motor Functions of Rats after Middle Cerebral Artery Occlusion.

There are numerous publications demonstrating that plant polyphenols can reduce oxidative stress and inflammatory processes in the brain. In the present study we have investigated the neuroprotective effect of plant extract isolated from the roots of L. gmelinii since it contains a rich source of polyphenols and other biologically active compounds. We have applied an oxidative and inflammatory model induced by NMDA, H2O2, and TNF-α in human primary neurons and astrocytes, and mouse cerebral endothelial cell (CECs) line in vitro. The levels of ROS generation, NADPH oxidase activation, P-selectin expression, and activity of ERK1/2 were evaluated by quantitative immunofluorescence analysis, confocal microscopy, and MAPK assay. In vivo, sensorimotor functions in rats with middle cerebral artery occlusion (MCAO) were assessed. In neurons NMDA induced overproduction of ROS, in astrocytes TNF-α initiated ROS generation, NADPH oxidase activation, and phosphorylation of ERK1/2. In CECs, the exposure by TNF-α induced oxidative stress and triggered the accumulation of P-selectin on the surface of the cells. In turn, pre-treatment of the cells with the extract of L. gmelinii suppressed oxidative stress in all cell types and pro-inflammatory responses in astrocytes and CECs. In vivo, the treatment with L. gmelinii extract improved motor activity in rats with MCAO.

Mortality of embryos, developmental disorders and changes in biochemical parameters in marsh frog (Rana ridibunda) tadpoles exposed to the water-soluble fraction of Kazakhstan crude oil and O-Xylene.

The effects of different concentrations of water-soluble fraction of crude oil (WSFO) from the Zhanazhol oil field (Aktobe region, Kazakhstan) and compared to o-xylene, prevalent in this oil, on growth and development of marsh frog (Rana ridibunda) were assessed. In subchronic experiments (7 d), a dose-related increase in mortality and incidence of deformities in embryos were observed. In chronic experiments (60 d; starting from the Gosner stage 26), a dose-dependent decrease in body weight, size and developmental delay by 3-4 stages were also detected. In addition, the content of lipid hyperoxide (LHO) and malondialdehyde (MDA), as well as activities of superoxide dismutase (SOD) and catalase (CAT) enzymes in liver of the tadpoles were determined at the end of chronic experiment. Exposure to 0.5 mg/L or 1.5 mg/L WSFO elevated the content of LHO by 76% and 86%, and MDA by 47% and 58% but decreased SOD activity by 26% and 49%, and CAT by 35% and 46%, respectively. A less pronounced adverse effect was found after chronic exposure to the same concentrations of o-xylene. In tadpole liver exposed to o-xylene levels of LHO was increased by 40% and 51%, MDA by 11% and 29%, while the activity of SOD was lowered by 18% and 41%, and CAT - by 13% and 37% in the 0.5 mg/L and 1.5 mg/L treatment groups, respectively. Data demonstrated the embryotoxic and teratogenic effects attributed to WSFO and o-xylene exposure which may involve oxidative stress mechanisms.

Chromosomal instability in rodents caused by pollution from Baikonur cosmodrome.

An assessment of the health status of ecosystems exposed to man-made pollution is a vital issue for many countries. Particularly it concerns the consequences of contamination caused by the activity of the space industry. Each rocket launch is accompanied by the introduction of parts of the rocket propellant into the environment. This study aims to scrutinize the effect of the components of rocket fuel on the induction of lipid peroxidation and chromosomal aberrations on rodents inhabiting the area exposed to pollution from Baikonur cosmodrome. The results showed the increase of the level of lipid hydroperoxide and malondialdehyde in the livers of Citellus pygmaeus Pallas and Mus musculus L., which indicates an augmentation of free radical activity and DNA damage. The cytogenetic analysis of bone marrow cells revealed that the frequency of chromosomal aberrations was a few times higher in the rodents from contaminated territory. The signs of oxidative stress and high level of chromosomal aberrations indicate the environmental impact of the cosmodrome, and its possible toxic and mutagenic effects on ecosystems.

Whole ovine ovaries as a model for human: perfusion with cryoprotectants in vivo and in vitro.

These experiments were performed to test the perfusion of ovine as a model for human ovaries by cryoprotectants in vivo at high temperature when the permeability of capillaries is high and when blood is insensibly replaced by the solution of cryoprotectants. By our hypothetical supposition, ovaries could be saturated by cryoprotectants before their surgical removal. The objective was to examine the effectiveness of perfusion of ovine ovaries with vascular pedicle in vivo and in vitro. Arteria ovarica was cannuled and ovaries were perfused by Leibovitz L-15 medium + 100 IU/mL heparin + 5% bovine calf serum + 6% dimethyl sulfoxide + 6% ethylene glycol + 0.15 M sucrose + Indian ink in vivo and in vitro. In the first and second cycle of experiments, ovaries (n = 13 and n = 23) were perfused in vivo and in vitro, respectively, during 60 min with the rate of perfusion 50 mL/h (0.8 mL/min). It was established with in vivo perfusion that only about 10% of ovarian tissues were perfused due to an appearance of multiple anastomoses when the perfusion medium goes from arteria ovarica to arteria uterina without inflow into the ovaries. It was concluded that in vitro perfusion of ovine intact ovaries with vascular pedicle by freezing medium is more effective than this manipulation performed in vivo.

Role of ROS in Aß42 Mediated Activation of Cerebral Endothelial Cells.

INTRODUCTION: There is substantial evidence that the deposition of aggregated amyloid-beta peptide (Aß) in brain parenchyma and brain vessels is the main cause of neuronal dysfunction and death in Alzheimer's disease (AD). Aß exhibits multiple cytotoxic effects on neurons and glial cells and causes dysfunction of the blood brain barrier (BBB). In AD brains, an increased deposition of Aß in the cerebral vasculature has been found to be correlated with increased transmigration of blood-borne inflammatory cells and neurovascular inflammation. However, regulatory mediators of these processes remain to be elucidated. In this study, we examined the role of ROS in actin polymerization and expression of adhesion molecules (P-selectin) on the surface of the cerebral endothelial cells (CECs) that are activated by Aß42. MATERIALS AND METHODS: Mouse BEnd3 line (ATCC) was used in this research. BEnd3 cells respond to Aß treatment similarly to human primary CECs and are a common model to investigate CECs' function. We used immortalized bEnd3 cells as the following: controls; cells incubated with Aß42 for 10, 30, and 60 minutes; cells incubated with 30 mM of antioxidant N-acetylcysteine (NAC) for 1 hr; and, cells pre-treated with NAC followed by Aß42 exposure. We measured DHE fluorescence to investigate intracellular ROS production. Immunofluorescent microscopy of anti-P-selectin and oregon green phalloidin was used to quantify the surface P-selectin expression and actin polymerization, and Western blot analysis was used to analyze total P-selectin expression. RESULTS: The results of this study have demonstrated a significant time-dependent ROS accumulation after 10 minutes, 30 minutes, and 60 minutes of Aß42 treatment, while Aß42 stimulated ROS production in CECs was attenuated by pre-treatment with the NAC antioxidant. We also found that Aß42 increased P-selectin fluorescence at the surface of bEnd3 cells in a time dependent manner in parallel to ROS elevation. However, total expression levels of P-selectin were not changed following exposure to Aß42. Pretreatment with NAC attenuated Aß42 induced P-selectin localization, while NAC alone did not significantly affect P selectin localization. As a positive control, H2O2 also increased P-selectin expression on the cell surface, which peaked after 30 minutes of H2O2 treatment. Exposure of CECs with Aß42 promoted actin polymerization, which peaked after 10 minutes of Aß42 treatment, while no significant increase of F-actin intensity was observed when cells were pre-treated with NAC. H2O2 was able to mimic Aß42 induced oxidative stress, causing increased actin polymerization with similar timing. CONCLUSIONS: The results of our study have indicated that Aß42 induced accumulation of P-selectin on the surface of bEnd3 cells and promoted actin polymerization, and all these events were correlated with ROS generation. The rapid post-translational cell signaling response mediated by ROS may well represent an important physiological trigger of the microvascular inflammatory responses in AD and requires further investigations.

Ectopic Liver Tissue Formation in Rats with Induced Liver Fibrosis.

INTRODUCTION: The possible alternative approach to whole-organ transplantation is a cell-based therapy, which can also be used as a "bridge" to liver transplantation. However, morphological and functional changes in the liver of patients suffering from chronic liver fibrosis and cirrhosis restrict the effectiveness of direct cell transplantation. Therefore, extra hepatic sites for cell transplantation, including the spleen, pancreas, peritoneal cavity, and subrenal capsule, could be a useful therapeutic approach for compensation of liver functions. However, a method of transplantation of hepatocytes into ectopic sites is needed to improve hepatocyte engraftment. Previously published data has demonstrated that mouse lymph nodes can support the engraftment and proliferation of hepatocytes as ES and rescue Fah mice from lethal liver failure. Thus, the aim of the study was to evaluate the engraftment of i.p. injected allogeneic hepatocytes into extra hepatic sites in albino rats with chemically induced liver fibrosis (LF). MATERIALS AND METHODS: Albino rats were randomly divided into 4 groups: (1) intact group (n = 18); (2) rats with induced LF (n = 18); (3) rats with induced LF and transplanted with hepatocytes (n = 18); (4) as a control, rats were treated with cyclosporine A only (n = 18). In order to prevent an immune response, groups 2 and 3 were subjected to immunosuppression by cyclosporine A (25 mg/kg per day). LF was induced using N-nitrosodimethylamine (NDMA), i.p., 10 mg/kg, three times a week for 4 weeks and confirmed by histological analysis of the liver samples. Hepatocytes transplantation (HT) was performed two days after NDMA exposure cessation by i.p. injection of 5×106 freshly isolated allogeneic hepatocytes. Liver function was assessed by quantifying blood biochemical parameters (ALT, AST, GGT, total protein, bilirubin, and albumin) at 1 week, 1 month, and 2 months after hepatocytes transplantation (HT). To confirm a hepatocytes' engraftment, we conducted immunohistochemical staining against HepPar1. RESULTS: We observed a 30% mortality rate among rats with LF within 1 week after NDMA exposure cessation, while 100% of animals with HT survived. ALT, AST, and GGT activities and bilirubin levels were markedly elevated in blood samples of LF rats compared to the control animals. However, HT significantly improved ALT, AST, and GGT activity as well as bilirubin levels. We also observed decreased levels of total protein and albumin in the blood serum of rats with LF, while HT normalized these parameters. At the same time, we have not detected any statistical differences of the studied parameters in the group 4, which was treated with Cyclosporine A only, compared with the intact animals. HepPar1 immunohistochemical staining of the different tissue sections demonstrated the presence of engrafted hepatocytes, mainly within enlarged Peyer's patches (aggregated lymphoid nodules in the lowest portion of the small intestine). CONCLUSION: The results of our study provide evidence that HT improves animal survival and liver functions. One potential reason for these results is that ectopic hepatic mass inside the Peyer's patches can rescue rats from liver failure.

Effects of Amyloid Beta Peptide on Neurovascular Cells.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder, which is characterized by the accumulation of amyloid plaques and neurofibrillary tangles in specific regions of the brain, accompanied by impairment of the neurons, and progressive deterioration of cognition and memory of affected individuals. Although the cause and progression of AD are still not well understood, the amyloid hypothesis is dominant and widely accepted. According to this hypothesis, an increased deposition of amyloid-ß peptide (Aß) in the brain is the main cause of the AD's onset and progression. There is increasing body of evidence that blood-brain barrier (BBB) dysfunction plays an important role in the development of AD, and may even precede neuron degeneration in AD brain. In the early stage of AD, microvasculature deficiencies, inflammatory reactions, surrounding the cerebral vasculature and endothelial dysfunctions are commonly observed. Continuous neurovascular degeneration and accumulation of Aß on blood vessels resulting in cerebral amyloid angiopathy is associated with further progression of the disease and cognitive decline. However, little is known about molecular mechanisms that underlie Aß induced damage of neurovascular cells. In this regards, this review is aimed to address how Aß impacts the cerebral endothelium. Understanding the cellular pathways triggered by Aß leading to alterations in cerebral endothelial cells structure and functions would provide insights into the mechanism of BBB dysfunction and inflammatory processes in Alzheimer's, and may offer new approaches for prevention and treatment strategies for AD.

Assessment of the mutagenic effect of 1,1-dimethyl hydrazine.

The mutagenic effect of the rocket fuel 1,1-dimethyl hydrazine has been studied experimentally and compared to the well-recognized mutagene N-nitroso dimethylamine. The manifestation of the effect for both compounds was disclosed through a significant increase in the chromosome aberration frequency in the bone marrow cells of intoxicated rats. The levels of chromosome aberrations induced by 1,1-dimetyl hydrazine were studied following both single (1h) and repeated doses (daily for 10 consecutive days) by inhalation (205-1028mg/m(3)) and gavage (5.4-26.8mg/kg) administration, respectively. For comparison N-nitroso dimethylamine were administered by inhalation (2h/daily for 10 consecutive days) and by gavage in concentrations of 2.4-48mg/m(3) and 1-30mg/kg, respectively. A clear dependence of concentration as well of time was disclosed. The BenchMark Dose approach was employed to derive guideline doses for the two compounds, the implications towards human health being discussed.