Gislaine Garcia Pelosi

Age-Related Changes in the Global DNA Methylation Profile of Leukocytes Are Linked to Nutrition but Are Not Associated with the MTHFR C677T Genotype or to Functional Capacities

Global DNA methylation of peripheral blood leukocytes has been recently proposed as a potential biomarker for disease risk. However, the amplitude of the changes in DNA methylation associated with normal aging and the impacts of environmental changes on this variation are still unclear. In this context, we evaluated the association of global DNA methylation with nutritional habits, tobacco smoking, body mass index (BMI), clinical laboratory parameters, polymorphism C677T MTHFR, functional cognition and the daily practice of physical activity in a cancer-free older population. Leukocyte global DNA methylation from 126 older individuals was quantified using a high-throughput ELISA-based method. Global DNA hypomethylation was observed in older individuals when compared to a younger population (p = 0.0469), confirming changes in DNA methylation in the aging process. Furthermore, the methylation profile of elders was correlated with the daily ingestion of carbohydrates (p = 0.0494), lipids (p = 0.0494), vitamin B6 (p = 0.0421), magnesium (p = 0.0302), and also to the serum levels of total protein (p = 0.0004), alpha 2 globulin (p = 0.0013) and albumin (p = 0.0015). No statistically significant difference was observed when global DNA methylation were stratified according to C677T MTHFR genotypes (p = 0.7200), BMI (p = 0.1170), smoking habit (p = 0.4382), physical activity in daily life (p = 0.8492), scored cognitive function (p = 0.7229) or depression state (p = 0.8301). Our data indicate that age-related variations in the global DNA methylation profile of leukocytes might be modulated by the daily intake of carbohydrates, lipids, vitamin B6, and magnesium and be associated with serum protein levels, however it is independent of C677T MTHFR genotype and not correlated with BMI, smoking habit, cognitive function or the routine physical activities.

Acute stress affects the global DNA methylation profile in rat brain: Modulation by physical exercise

The vulnerability of epigenetic marks of brain cells to environmental stimuli and its implication for health have been recently debated. Thus, we used the rat model of acute restraint stress (ARS) to evaluate the impact of stress on the global DNA methylation and on the expression of the Dnmt1 and Bdnf genes of hippocampus, cortex, hypothalamus and periaqueductal gray (PAG). Furthermore, we verified the potential of physical exercise to modulate epigenetic responses evoked by ARS. Sedentary male Wistar rats were submitted to ARS at the 75th postnatal day (PND), whereas animals from a physically active group were previously submitted to swimming sessions (35-74th PND) and to ARS at the 75th PND. Global DNA methylation profile was quantified using an ELISA-based method and the quantitative expression of the Dnmt1 and Bdnf genes was evaluated by real-time PCR. ARS induced a decrease in global DNA methylation in hippocampus, cortex and PAG of sedentary animals and an increased expression of Bdnf in PAG. No change in DNA methylation was associated with ARS in the exercised animals, although it was associated with abnormal expression of Dnmt1 and Bdnf in cortex, hypothalamus and PAG. Our data reveal that ARS evokes adaptive changes in global DNA methylation of rat brain that are independent of the expression of the Dnmt1 gene but might be linked to abnormal expression of the Bdnf gene in the PAG. Furthermore, our evidence indicates that physical exercise has the potential to modulate changes in DNA methylation and gene expression consequent to ARS.

Dorsal periaqueductal gray area synapses modulate baroreflex in unanesthetized rats

The dorsal portion of the periaqueductal gray area (dPAG) is involved in behavioral and cardiovascular control. We report the effect of acute and reversible dPAG blockade by local microinjection of either lidocaine or CoCl2 on the baroreflex response of unanesthetized rats. Acute and reversible blockade evoked by lidocaine microinjection into the dPAG did not affect the bradycardic response to mean arterial pressure (MAP) increases evoked by i.v. infusion of phenylephrine. However, lidocaine increased baroreflex gain and tachycardic reflex in response to MAP decreases evoked by i.v. infusion of sodium nitroprusside, thus suggesting an action on the sympathetic component of the baroreflex. The effects of dPAG synapses blockade caused by CoCl2 were similar to those observed after lidocaine microinjection. CoCl2 microinjection also increased baroreflex gain and tachycardiac responses to MAP decreases without affecting the parasympathetic baroreflex component. In conclusion, our data point to a dPAG tonic inhibitory involvement in baroreflex control, specifically modulating the sympathetic baroreflex component. Temporary dPAG ablation by local microinjection of lidocaine increased the sympathetic baroreflex component. Because CoCl2 microinjection had similar effects on the baroreflex, this modulation involves local synaptic neurotransmission within the dPAG.

Maternal exposure to fluoxetine during gestation and lactation affects the DNA methylation programming of rat's offspring: Modulation by folic acid supplementation

Fluoxetine is an antidepressant that has been largely used for treatment of depression in pregnancy. In the present study we evaluated the effects of the exposure to fluoxetine during gestation and lactation on DNA methylation of rat brain regions. Female Wistar rats were treated with 5mg/kg of fluoxetine during pregnancy and lactation. In order to assess the effects of fluoxetine in the context of maternal folic acid supplementation we performed an additional combined treatment composed by folic acid (8 mg/kg/day) and fluoxetine (5 mg/kg/day). On the postnatal day 22, male rats were euthanized and hippocampus, cortex, hypothalamus, and periaqueductal gray area were removed. Global DNA methylation was quantified using a high-throughput ELISA-based method. Neurofunctional changes were addressed using validated behavioral tests: hot plate, elevated plus maze and open field. A decrease in the global DNA methylation profile of hippocampus was associated to the exposure to fluoxetine, whereas an increase in methylation was observed in cortex. The combined treatment induced an increase in the methylation of hippocampus indicating the potential of folic acid to modulate this epigenetic alteration. Increase in the latency to the thermal nociceptive response was observed in animals exposed to fluoxetine whereas this effect was abolished in animals from the combined treatment. In summary we demonstrated that exposure to fluoxetine during gestation and lactation affect the DNA methylation of brain and the nociceptive response of rats. Furthermore our data reveal the potential of folic acid to modulate epigenetic and functional changes induced by early exposure to fluoxetine.

Fluoxetine exposure during pregnancy and lactation: Effects on acute stress response and behavior in the novelty-suppressed feeding are age and gender-dependent in rats

Fluoxetine (FLX) is commonly used to treat anxiety and depressive disorders in pregnant women. Since FLX crosses the placenta and is excreted in milk, maternal treatment with this antidepressant may expose the fetus and neonate to increased levels of serotonin (5-HT). Long-term behavioral abnormalities have been reported in rodents exposed to higher levels of 5-HT during neurodevelopment. In this study we evaluated if maternal exposure to FLX during pregnancy and lactation would result in behavioral and/or stress response disruption in adolescent and adult rats. Our results indicate that exposure to FLX influenced restraint stress-induced Fos expression in the amygdala in a gender and age-specific manner. In male animals, a decreased expression was observed in the basolateral amygdala at adolescence and adulthood; whereas at adulthood, a decrease was also observed in the medial amygdala. A lack of FLX exposure effect was observed in females and also in the paraventricular nucleus of both genders. Regarding the behavioral evaluation, FLX exposure did not induce anhedonia in the sucrose preference test but decreased the latency to feed of both male and female adolescent rats evaluated in the novelty-suppressed feeding test. In conclusion, FLX exposure during pregnancy and lactation decreases acute amygdalar stress response to a psychological stressor in males (adolescents and adults) as well as influences the behavior of adolescents (males and females) in a model that evaluates anxiety and/or depressive-like behavior. Even though FLX seems to be a developmental neurotoxicant, the translation of these findings to human safe assessment remains to be determined since it is recognized that not treating a pregnant or lactating woman may also impact negatively the development of the descendants.

Cardiovascular effects of noradrenaline microinjected into the dorsal periaqueductal gray area of unanaesthetized rats

The periaqueductal grey area (PAG) is a mesencephalic region that is involved in the modulation of cardiovascular changes associated with behavioural responses. Among the neurotransmitters present in the PAG, noradrenaline (NA) is also known to be involved in central nervous system cardiovascular regulation. In the present study we report the cardiovascular effects of the microinjection of NA into the dorsal portion of the PAG (dPAG) of unanaesthetized rats and the peripheral mechanism involved in their mediation. Injection of NA in the dPAG of unanaesthetized rats evoked a dose‐dependent pressor response accompanied by bradycardia. The magnitude of the pressor responses was higher at more rostral sites in the dPAG and decreased when NA was injected into the caudal portion of the dPAG. The responses to NA were markedly reduced in urethane‐anaesthetized rats. The pressor response was potentiated by i.v. pretreatment with the ganglion blocker pentolinium and blocked by i.v. pretreatment with the vasopressin antagonist dTyr(CH2)5(Me)AVP. The results suggest that activation of noradrenergic receptors within the dPAG can evoke pressor responses, which are mediated by acute vasopressin release.

The paraventricular nucleus of hypothalamus mediates the pressor response to noradrenergic stimulation of the medial prefrontal cortex in unanesthetized rats

The medial prefrontal cortex (MPFC) is a structure that is also involved in cardiovascular modulation. The injection of norepinephrine (NE) into the prelimbic (PL) area of the MPFC of unanesthetized rats evokes a pressor response which is mediated by acute vasopressin release. Vasopressin is synthesized by magnocellular cells of the paraventricular (PVN) and supraoptic nucleus (SON) of the hypothalamus. In the present study, we endeavored to determine which vasopressin-synthesizing hypothalamic nucleus is involved in the pressor pathway activated after NE injection into the PL area of the MPFC. We report here that lidocaine microinjection into the SON did not change the pressor response evoked by NE injection into the PL. However, the response to NE was blocked by prior injection of lidocaine or CoCl(2) into the PVN, indicating that this area is responsible for the mediation of this pressor response. A neuroanatomic experiment in which the neuronal tracer biotinylated dextran amine (BDA) was microinjected into the MPFC showed a lack of axons or neuronal cell bodies in the PVN, indicating that there are no direct connections between the PL area of the MPFC and the PVN. The results suggest that the PVN is involved in the mediation of the pressor response to NE in the PL area and that this pathway must relay in other brain structures before reaching the PVN.

The paraventricular nucleus of the hypothalamus is involved in cardiovascular responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of cardiovascular control. Stimulation of the PVN evokes changes in blood pressure and heart rate. Additionally, this brain area is connected to several limbic structures implicated in behavioral control, as well as to forebrain and brainstem structures involved in cardiovascular control. This evidence indicates that the PVN may modulate cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint is an unavoidable stressor that evokes marked and sustained cardiovascular changes, which are characterized by elevated mean arterial pressure (MAP) and an intense heart rate (HR) increase. We report on the effect of inhibition of PVN synapses on MAP and HR responses evoked by acute restraint in rats. Bilateral microinjection of the nonspecific synaptic blocker cobalt (CoCl2, 1 mM/100 nl) into the PVN did not change the HR response or the initial peak of the MAP response to restraint stress, but reduced the area under the curve of the MAP response. Moreover, bilateral microinjection of cobalt in areas surrounding the PVN did not change the cardiovascular response to restraint. These results indicate that synapses in the PVN are involved in the neural pathway that controls blood pressure changes evoked by restraint.

Epigenetic vulnerability and the environmental influence on health

A growing body of evidence has drawn the attention of the scientific community by indicating the potential vulnerability to environmental changes of epigenetic mechanisms that control gene expression. Being critical components of normal development, the importance of epigenetic mechanisms for normal biology is illustrated by the fact that abnormal epigenetic patterns have increasingly been linked to the aetiology of various diseases including cancer, paediatric syndromes, autoimmune diseases, genetic disorders and even the molecular process of ageing. It is estimated that the degree of vulnerability to changes in epigenetic patterns is high during early embryonic development, a period of life in which epigenetic patterns are established and cell differentiation is intense. Moreover, increasing amounts of relevant data and information reveal that the environment might potentially impact on epigenetic patterns at every period of life. Within this context, in this study we will review the principles of epigenetic vulnerability to environmental changes, the impacts on development, the association with the origin of common diseases and also speculate about the potential of lifestyle changes to modulate epigenetic patterns and contribute to preventing common diseases.

Involvement of the periaqueductal gray in the hypotensive response evoked by l-glutamate microinjection in the lateral hypothalamus of unanesthetized rats

The lateral hypothalamus (LH) is involved in cardiovascular control. L-glutamate (L-glu) stimulation of the LH of unanesthetized rats evoked hypotensive responses without significant heart rate changes. The neuronal pathway that mediates this response is unknown. There is evidence that the periaqueductal gray (PAG) is involved in the mediation of hypotensive responses evoked by electrical stimulation of the LH. In the present study, we attempted to verify the effect of an acute and reversible pharmacological ablation of the PAG with lidocaine or CoCl(2) on the hypotensive response caused by L-glu injection in the LH of unanesthetized rats. Microinjection of the local anesthetic lidocaine or the unspecific synaptic blocker CoCl(2) in the PAG significantly attenuated the hypotensive effects of L-glu stimulation of the LH, indicating the involvement of local synapses within the PAG in the hypotensive pathway activated by LH glutamatergic receptors. Microinjection of the neuronal tracer biotinylated dextran amine (BDA) in the PAG labeled neuronal cell bodies in the LH, indicating the existence of direct connections between these areas. In conclusion, the present results indicate that the hypotensive response evoked by L-glu stimulation of LH may involve a synaptic relay in the dorsal PAG.