Effects of photobiomodulation and swimming on gene expression in rats with the tibialis anterior muscle injury.

The aim of the present study was to evaluate the effects of photobiomodulation (low-level laser therapy (LLLT)) and aquatic exercise on the expression of genes related to muscle regeneration in rats. Wistar rats were divided into five groups: control group (n = 15), non-treated injury group (n = 15), injury+LLLT group (n = 15), injury+aquatic exercise group (n = 15), and injury+LLLT+aquatic exercise group (n = 15). Cryoinjury was performed on the belly of the tibialis anterior (TA) muscle. LLLT was performed daily with an AlGaAs laser (830 nm; beam spot of 0.0324 cm2, output power of 100 mW, energy density of 180 J/cm2, and 58-s exposure time). Animals were euthanized at 7, 14, and 21 days. The TA muscles were removed for gene expression analysis of TGF-ß, Myogenin, and MyoD. The results were statistically analyzed at a significance level of 5%. The cryoinjury increased the expression of genes related to muscle regeneration-MyoD, Myogenin, and TGF-ß-compared to the control group (p < 0.05); the photobiomodulation increased the expression of these genes at day 7 (p < 0.05), decreasing until day 21; and the aquatic exercise increases the expression of the three genes over time. When the two treatments were combined, the expression of the analyzed genes also increased over time. In summary, the results of our study suggest that photobiomodulation (LLLT), when applied alone in cryoinjury, is able to increase the gene expression of MyoD, Myogenin, and TGF-ß at the acute phase, while when combined with aquatic exercises, there is an increase in expression of these genes specially at the long-term treatment.

Dietary Ingestion of Calories and Micronutrients Modulates the DNA Methylation Profile of Leukocytes from Older Individuals.

OBJECTIVE: Several lines of evidence from the last decade support the connection between nutrition and epigenetic mechanisms. In the present study we evaluated the impact of the daily dietary intake of calories and the micronutrients vitamin A, D, B1, B2, B5, C, E, copper, calcium, phosphorus, iron, iodine, selenium, manganese, potassium and sodium on the global DNA methylation profile of blood cells from older individuals. RESEARCH METHODS AND PROCEDURES: The study enrolled 126 physically independent elderly of both sexes (60 men and 66 women). For the molecular analysis, DNA samples were extracted from leukocytes and global DNA methylation was evaluated using a high throughput Elisa-based method. Correlations between global DNA methylation and the daily intake of calorie or micronutrients were evaluated using Prism5 GraphPad Software. RESULTS: A statistically significant correlation was observed between global DNA methylation and the daily caloric value (p=0.019, r=-0.21), and the intake of vitamin A (p=0.03, r=-0.18), Vitamin E (p=0.027, r=-0.20) and copper (p=0.04, r=-0.18). No correlation was observed between global DNA methylation and the daily intake of vitamin D, B1, B2, B5, C, calcium, phosphorus, iron, iodine, selenium, manganese and potassium (p>0.05). CONCLUSION: Our data demonstrate that the daily intake of calories or the micronutrients vitamin A, vitamin E and copper can potentially modulate the global DNA methylation profile of leukocytes in older adults and corroborate the notion of nutritional influences on epigenetic mechanisms.

Maternal exposure to fluoxetine during gestation and lactation induces long lasting changes in the DNA methylation profile of offspring's brain and affects the social interaction of rat.

Fluoxetine (FLX) is an antidepressant from the selective serotonin reuptake inhibitor class that has largely been used for the treatment of depression in pregnancy. However, increasing evidences have indicated the potential of early maternal exposure to FLX to induce molecular and neuro functional effects on the offspring. In the present study we evaluated possible long lasting impacts of the maternal exposure to FLX during gestation and lactation. Female Wistar rats were gavaged with 5 mg/kg of FLX during the period that comprehends the first day of pregnancy (PD0) and the last day of lactation (LD21) (Group FLX). Control group (CTL) received a proportional volume of water. On the postnatal day 75 (PND75), male rats were euthanized and hippocampus, cortex, hypothalamus, and periaqueductal gray area (PAG) were removed. Global DNA methylation was quantified using a high-throughput ELISA-based method. In order to address neuro functional changes animals (PND75) were evaluated in the elevated plus maze and social interaction tests as well as submitted to repeated restraint stress. An increase in the global DNA methylation profile of hippocampus (p = 0.0399) was associated with the early exposure to FLX, whereas no significant change was observed in the hypothalamus (p = 0.6556), cortex (p = 0.9402) or PAG (p = 0.3822). Furthermore, early exposure to FLX was also associated with a reduction in the social interaction time (p = 0.0084) and to a decreased in the plasma corticosterone level when animals were submitted to the restraint stress (p < 0.0001). No significant change in the elevated plus maze test was associated with the early exposure to FLX. In summary, our data demonstrate that maternal exposure to FLX during gestation and lactation results in a long lasting impact on the DNA methylation of hippocampus, and affects the social behavior and the corticosterone response to stress.

Effects of chronic restraint stress on the global DNA methylation profile of rat lung cells: Modulation by physical exercise.

The potential of behavioral stress to affect epigenetic mechanisms of non-encephalic tissues is still underestimated. In the present study we evaluated the effects of chronic behavioral stress on the DNA methylation profile of rat lung cells. Furthermore, we evaluated the potential of physical exercise to modulate the changes evoked by behavioral stress in lung cells. Male Wistar rats were divided into four experimental groups: (1) animals submitted to chronic restraint stress (CRS) (ST group) during the period of the 67th-80th postnatal day (PND); (2) animals submitted to physical exercise (EX group) during the 53rd-79th PND; (3) animals submitted to swimming during the 53rd-79th PND and to CRS during the 67th-80th PND (EX-ST group); and (4) animals not submitted to stress or swimming protocols (CTL). Global DNA methylation was quantified using an ELISA-based approach and gene expression was evaluated by real time PCR. A decreased global DNA methylation profile was observed in the ST group, however physical exercise demonstrated protection of lung cells from this stress-related hypomethylation. Increased expression of the Dnmt1 gene was evidenced in the ST group, whereas physical exercise was shown to protect lung cells from this stress-related effect in the EX-ST group. Comparative analysis of the ST and EX groups revealed opposite effects on the expression of Dnmt3a and Dnmt3b; however, a stress-related increase in expression of Dnmt3a and Dnmt3b was not seen in the EX-ST group. Our data showed that behavioral stress induced significant changes in the DNA methylation profile of rat lung cells and that this could be modulated by physical exercise.

Physical exercise affects the epigenetic programming of rat brain and modulates the adaptive response evoked by repeated restraint stress.

Epigenetics has recently been linked to molecular adaptive responses evoked by physical exercise and stress. Herein we evaluated the effects of physical exercise on global DNA methylation and expression of the Dnmt1 gene in the rat brain and also verified its potential to modulate responses evoked by repeated restraint stress (RRS). Wistar rats were classified into the following experimental groups: (1) physically active (EX): animals submitted to swimming during postnatal days 53-78 (PND); (2) stress (ST): animals submitted to RRS during 75-79PND; (3) exercise-stress (EX-ST): animals submitted to swimming during 53-78PND and to RRS during 75-79PND, and (4) control (CTL): animals that were not submitted to intervention. Samples from the hippocampus, cortex and hypothalamus were obtained at 79PND. The global DNA methylation profile was assessed using an ELISA-based method and the expression of Dnmt1 was evaluated by real-time PCR. Significantly increased methylation was observed in the hypothalamus of animals from the EX group in comparison to CTL. Comparative analysis involving the EX-ST and ST groups revealed increased global DNA methylation in the hippocampus, cortex, and hypothalamus of EX-ST, indicating the potential of physical exercise in modulating the responses evoked by RRS. Furthermore, decreased expression of the Dnmt1 gene was observed in the hippocampus and hypothalamus of animals from the EX-ST group. In summary, our data indicate that physical exercise affects DNA methylation of the hypothalamus and might modulate epigenetic responses evoked by RRS in the hippocampus, cortex, and hypothalamus.

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.

Angiotensin (5-8) modulates nociception at the rat periaqueductal gray via the NO-sGC pathway and an endogenous opioid.

Angiotensins (Angs) modulate blood pressure, hydro-electrolyte composition, and antinociception. Although Ang (5-8) has generally been considered to be inactive, we show here that Ang (5-8) was the smallest Ang to elicit dose-dependent responses and receptor-mediated antinociception in the rat ventrolateral periaqueductal gray matter (vlPAG). Ang (5-8) antinociception seems to be selective, because it did not alter blood pressure or act on vascular or intestinal smooth muscle cells. The non-selective Ang-receptor (Ang-R) antagonist saralasin blocked Ang (5-8) antinociception, but selective antagonists of Ang-R types I, II, IV, and Mas did not, suggesting that Ang (5-8) may act via an unknown receptor. Endopeptidase EP 24.11 and amastatin-sensitive aminopeptidase from the vlPAG catalyzed the synthesis (from Ang II or Ang III) and inactivation of Ang (5-8), respectively. Selective inhibitors of neuronal-nitric oxide (NO) synthase, soluble guanylyl cyclase (sGC) and a non-selective opioid receptor (opioid-R) inhibitor blocked Ang (5-8)-induced antinociception. In conclusion, Ang (5-8) is a new member of the Ang family that selectively and strongly modulates antinociception via NO-sGC and endogenous opioid in the vlPAG.

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.