Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model.

Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 - on E20 and P20, Hprt1, Actb, and Pgk1 - on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.

Epigenetic Mechanisms Involved in the Effects of Maternal Hyperhomocysteinemia on the Functional State of Placenta and Nervous System Plasticity in the Offspring.

According to modern view, susceptibility to diseases, specifically to cognitive and neuropsychiatric disorders, can form during embryonic development. Adverse factors affecting mother during the pregnancy increase the risk of developing pathologies. Despite the association between elevated maternal blood homocysteine (Hcy) and fetal brain impairments, as well as cognitive deficits in the offspring, the role of brain plasticity in the development of these pathologies remains poorly studied. Here, we review the data on the negative impact of hyperhomocysteinemia (HHcy) on the neural plasticity, in particular, its possible influence on the offspring brain plasticity through epigenetic mechanisms, such as changes in intracellular methylation potential, activity of DNA methyltransferases, DNA methylation, histone modifications, and microRNA expression in brain cells. Since placenta plays a key role in the transport of nutrients and transmission of signals from mother to fetus, its dysfunction due to aberrant epigenetic regulation can affect the development of fetal CNS. The review also presents the data on the impact of maternal HHcy on the epigenetic regulation in the placenta. The data presented in the review are not only interesting from purely scientific point of view, but can help in understanding the role of HHcy and epigenetic mechanisms in the pathogenesis of diseases, such as pregnancy pathologies resulting in the delayed development of fetal brain, cognitive impairments in the offspring during childhood, and neuropsychiatric and neurodegenerative disorders later in life, as well as in the search for approaches for their prevention using neuroprotectors.

Imbalance of Angiogenic and Growth Factors in Placenta in Maternal Hyperhomocysteinemia.

Numerous studies have shown that various adverse factors of different nature and action mechanisms have similar negative influence on placental angiogenesis, resulting in insufficiency of placental blood supply. One of the risk factors for pregnancy complications with placental etiology is an increased level of homocysteine in the blood of pregnant women. However, the effect of hyperhomocysteinemia (HHcy) on the development of the placenta and, in particular, on the formation of its vascular network is at present poorly understood. The aim of this work was to study the effect of maternal HHcy on the expression of angiogenic and growth factors (VEGF-A, MMP-2, VEGF-B, BDNF, NGF), as well as their receptors (VEGFR-2, TrkB, p75NTR), in the rat placenta. The effects of HHcy were studied in the morphologically and functionally different maternal and fetal parts of the placenta on the 14th and 20th day of pregnancy. The maternal HHcy caused increase in the levels of oxidative stress and apoptosis markers accompanied by an imbalance of the studied angiogenic and growth factors in the maternal and/or fetal part of the placenta. The influence of maternal HHcy in most cases manifested in a decrease in the protein content (VEGF-A), enzymatic activity (MMP-2), gene expression (VEGFB, NGF, TRKB), and accumulation of precursor form (proBDNF) of the investigated factors. In some cases, the effects of HHcy differed depending on the placental part and stage of development. The influence of maternal HHcy on signaling pathways and processes controlled by the studied angiogenic and growth factors could lead to incomplete development of the placental vasculature and decrease in the placental transport, resulting in fetal growth restriction and impaired fetal brain development.

Neurotrophins: are they involved in immune tolerance in pregnancy?

In this review, an attempt was made to substantiate the possibility for neurotrophins to be involved in the development of immune tolerance based on data accumulated on neurotrophin content and receptor expression in the trophoblast and immune cells, in particular, in natural killer cells. Numerous research results are reviewed to show that the expression and localization of neurotrophins along with their high-affinity tyrosine kinase receptors and low-affinity p75NTR receptor in the mother-placenta-fetus system indicate the important role of neurotrophins as binding molecules in regulating the crosstalk between the nervous, endocrine, and immune systems in pregnancy. An imbalance between these systems can occur with tumor growth and pathological processes observed in pregnancy complications and fetal development anomalies.

Maternal Hyperhomocysteinemia Disturbs the Mechanisms of Embryonic Brain Development and Its Maturation in Early Postnatal Ontogenesis.

Maternal hyperhomocysteinemia causes the disruption of placental blood flow and can lead to serious disturbances in the formation of the offspring's brain. In the present study, the effects of prenatal hyperhomocysteinemia (PHHC) on the neuronal migration, neural tissue maturation, and the expression of signaling molecules in the rat fetal brain were described. Maternal hyperhomocysteinemia was induced in female rats by per os administration of 0.15% aqueous methionine solution in the period of days 4-21 of pregnancy. Behavioral tests revealed a delay in PHHC male pups maturing. Ultrastructure of both cortical and hippocampus tissue demonstrated the features of the developmental delay. PHHC was shown to disturb both generation and radial migration of neuroblasts into the cortical plate. Elevated Bdnf expression, together with changes in proBDNF/mBDNF balance, might affect neuronal cell viability, positioning, and maturation in PHHC pups. Reduced Kdr gene expression and the content of SEMA3E might lead to impaired brain development. In the brain tissue of E20 PHHC fetuses, the content of the procaspase-8 was decreased, and the activity level of the caspase-3 was increased; this may indicate the development of apoptosis. PHHC disturbs the mechanisms of early brain development leading to a delay in brain tissue maturation and formation of the motor reaction of pups.

Prenatal Hyperhomocysteinemia Induces Glial Activation and Alters Neuroinflammatory Marker Expression in Infant Rat Hippocampus.

Maternal hyperhomocysteinemia is one of the common complications of pregnancy that causes offspring cognitive deficits during postnatal development. In this study, we investigated the effect of prenatal hyperhomocysteinemia (PHHC) on inflammatory, glial activation, and neuronal cell death markers in the hippocampus of infant rats. Female Wistar rats received L-methionine (0.6 g/kg b.w.) by oral administration during pregnancy. On postnatal days 5 and 20, the offspring's hippocampus was removed to perform histological and biochemical studies. After PHHC, the offspring exhibited increased brain interleukin-1ß and interleukin-6 levels and glial activation, as well as reduced anti-inflammatory interleukin-10 level in the hippocampus. Additionally, the activity of acetylcholinesterase was increased in the hippocampus of the pups. Exposure to PHHC also resulted in the reduced number of neurons and disrupted neuronal ultrastructure. At the same time, no changes in the content and activity of caspase-3 were found in the hippocampus of the pups. In conclusion, our findings support the hypothesis that neuroinflammation and glial activation could be involved in altering the hippocampus cellular composition following PHHC, and these alterations could be associated with cognitive disorders later in life.

Prenatal Stress in Maternal Hyperhomocysteinemia: Impairments in the Fetal Nervous System Development and Placental Function.

The article presents current views on maternal hyperhomocysteinemia (HHcy) as an important factor causing prenatal stress and impaired nervous system development in fetuses and newborns in early ontogenesis, as well as complications in adulthood. Experimental data demonstrate that prenatal HHcy (PHHcy) affects the morphological maturation of the brain and activity of its neurotransmitter systems. Cognitive deficit observed in the offspring subjected to PHHcy in experimental studies can presumably cause the predisposition to various neurodegenerative diseases, as the role of maternal HHcy in the pathogenesis such diseases has been proven in clinical studies. The review also discusses molecular mechanisms of the HHcy neurotoxic action on the nervous system development in the prenatal and early postnatal periods, which include oxidative stress, apoptosis activation, changes in the DNA methylation patterns and microRNA levels, altered expression and processing of neurotrophins, and neuroinflammation induced by an increased production of pro-inflammatory cytokines. Special attention is given to the maternal HHcy impact on the placenta function and its possible contribution to the brain function impairments in the offspring. Published data suggest that some effects of PHHcy on the developing fetal brain can be due to the disturbances in the transport functions of the placenta resulting in an insufficient supply of nutrients necessary for the proper formation and functioning of brain structures.

Oxidative stress in serum and peripheral blood leukocytes in patients with different disease courses of multiple sclerosis.

BACKGROUND: The role of oxidative stress in patients with multiple sclerosis (MS) is poorly understood. OBJECTIVE: To investigate oxidative stress in serum and peripheral blood leukocytes in patients with different disease courses of MS. METHODS: Diene conjugate (DC) levels (a measure of lipid peroxidation), total antioxidative activity (AOA) and total antiradical activity (ARA) were measured in serum and peripheral blood leukocytes from 30 patients with benign relapsing remitting MS (BMS), 27 with secondary progressive MS (SPMS), 29 with primary progressive MS (PPMS), and 30 healthy controls. All MS patients were in a clinically stable phase. RESULTS: Serum DC levels were elevated in patients with BMS (p <0.05), SPMS (p <0.01) and PPMS (p <0.001). Serum total AOA and ARA were not different between MS patients and controls. Compared to controls, leukocyte DC levels were not different in each MS subgroup, but total ARA was elevated. There was a strong correlation, both in controls and MS patients, between leukocyte DC levels and leukocyte total ARA (p <0.0001) and leukocyte total AOA (p <0.0001). CONCLUSION: Oxidative stress occurs in progressive as well as benign MS. The finding that cells withstand oxidative stress, due to upregulated cellular antioxidant defence mechanisms, suggests that reactive oxygen species (ROS) formation in MS is not necessarily deleterious.

Effects of phentermine and phenformin on biomarkers of aging in rats.

BACKGROUND: Caloric restriction (CR) is the only treatment known to substantially prolong both average and maximal life span in experimental animals. Interventions that mimic certain effects of CR could be potential anti-aging treatments in humans. Drugs which reduce appetite (anorexiants) represent one class of candidate treatments. Agents that reduce the glucose utilization by the organism could also represent another class of candidate CR mimetics. OBJECTIVE: In our study, we addressed the following questions: (1) Does treatment with an anorexiant reduce caloric intake and body weight of experimental animals comparable to that caused by CR? (2) Does treatment with an antidiabetic agent influence caloric intake and body weight? (3) Does treatment with any of these drugs affect metabolic parameters of an organism in the way similar to that observed with CR? METHODS: One hundred and twenty 6-month-old female Wistar-derived LIO rats were randomly subdivided into four groups and exposed to: (1) ad libitum feeding with placebo (controls); (2) the antidiabetic drug phenformin (2 mg/kg); (3) the anorectic drug phentermine (1 mg/kg), and (4) the same amount of food as the group with the least food intake during the previous week (pair-fed controls). Food and water intake, body weight, and rectal temperature were measured weekly during 16 weeks. At the end of the 16th week of the experiment, serum levels of glucose, total beta-lipoprotein and pre-beta-lipoprotein fractions, cholesterol, triglycerides, insulin, total triiodothyronine, and free thyroxine were estimated. The contents of diene conjugates and Schiff's bases, total antioxidant activity, the activities of Cu/Zn superoxide dismutase, glutathione S-transferase, and glutathione peroxidase, and the generation of reactive oxygen species (ROS) were studied in brain and liver homogenates and in the serum. RESULTS: The controls exposed to pair feeding had a significantly reduced food consumption (about 20%) as compared with the ad libitum fed controls and thus exhibited a moderate CR. Treatment with phentermine reduced the caloric intake by about 12% as compared with the ad libitum fed controls. Body weight and water intake in this group were only slightly decreased (by about 2 and 5%, respectively) as compared with the controls. The mean rectal temperature in the phentermine group (38 degrees C) was significantly higher than in the ad libitum fed (37.8 degrees C) and pair-fed (37.6 degrees C) controls. Treatment with phentermine also resulted in significantly reduced ROS levels in all tissues studied, while the highest ROS production was found in ad libitum (blood serum) and pair-fed (brain) controls. Treatment with phenformin did not significantly influence food and water consumption, body weight, and temperature when compared with the ad libitum fed controls. Rats treated with this antidiabetic drug showed intermediate values of ROS generation. Differences among the groups in total antioxidant activity were not obvious. CONCLUSIONS: Treatment with phentermine reduces caloric intake slightly less than is commonly observed in CR studies. CR due to forcibly reduced feeding and CR due to substance-suppressed appetite appear to act through different metabolic mechanisms and thus may affect aging and longevity in different ways.

Peripheral blood leukocyte NO production in MS patients with a benign vs progressive course.

BACKGROUND: Nitric oxide (NO) may play a role in tissue destruction and axonal degeneration in multiple sclerosis (MS). OBJECTIVE: To investigate NO production by peripheral blood leukocytes (PBL) in patients with a benign and progressive course of MS. METHODS: PBL were isolated from 25 patients with a benign course of MS (BMS), 33 with secondary progressive MS (SPMS), 21 with primary progressive MS (PPMS), and 29 healthy individuals. Leukocyte supernatants were assayed for nitrite concentration, which is an index of NO generation, using the Griess reaction. Serum levels of tumor necrosis factor (TNF)alpha and interleukin (IL)-12 were measured using ELISA. RESULTS: Compared to healthy controls, nitrite concentrations were higher in patients with BMS (p < 0.001), SPMS (p < 0.001), and PPMS (p < 0.05). There were no significant differences among the three clinical subgroups of MS. There was a correlation between nitrite concentrations and serum levels of IL-12 (p = 0.04), but not of TNFalpha. CONCLUSION: Increased NO production by PBL in patients with MS is independent of the disease course.