Tiago Marcon dos Santos

Vitamin D 3 Reverses the Hippocampal Cytoskeleton Imbalance But Not Memory Deficits Caused by Ovariectomy in Adult Wistar Rats

The objective of study was to investigate changes caused by ovariectomy (OVX) on aversive and non-aversive memories, as well as on cytoskeleton phosphorylating system and on vitamin D receptor (VDR) immunocontent in hippocampus. The neuroprotective role of vitamin D was also investigated. Ninety-day-old female Wistar rats were divided into four groups: SHAM, OVX, VITAMIN D and OVXxa0+xa0VITAMIN D; 30xa0days after the OVX, vitamin D supplementation (500xa0IU/kg), by gavage, for 30xa0days was started. Results showed that OVX impaired short-term and long-term recognition, and long-term aversive memories. OVX altered hippocampal cytoskeleton phosphorylating system, evidenced by the hyperphosphorylation of glial fibrillary acidic protein (GFAP), low molecular weight neurofilament subunit (NFL), medium molecular weight neurofilament subunit (NFM) and high molecular weight neurofilament subunit (NFH), and increased the immunocontent of c-Jun N-terminal protein kinases (JNK), Ca2+/calmodulin-dependent protein kinase II (PKCaMII) and of the sites phosphorylated lysine–serine–proline (KSP) repeats, Ser55 and Ser57. Vitamin D reversed the effects caused by OVX on cytoskeleton in hippocampus, but it was not able to reverse the effects on memory.

Vitamin D Supplementation Reverses DNA Damage and Telomeres Shortening Caused by Ovariectomy in Hippocampus of Wistar Rats

The aim of this study was to investigate the effect of ovariectomy (OVX), a surgical model of menopause, and/or vitamin D (VIT D) supplementation on oxidative status, DNA damage, and telomere length in hippocampus of rats at two ages. Ninety-day-old (adult) or 180-day-old (older) female Wistar rats were divided into four groups: SHAM, OVX, VIT D, and OVXu2009+u2009VIT D. Thirty days after OVX, rats were supplemented with VIT D (500xa0IU/kg) by gavage, for a period of 30xa0days. Results showed that OVX altered antioxidant enzymes, increasing the activities of catalase in adult rats and superoxide dismutase in older rats. VIT D per se increased the activities of catalase and superoxide dismutase in older rats, but not in adult rats. VIT D supplementation to OVX (OVXu2009+u2009VIT D) rats did not reverse the effect of OVX on catalase in adult rats, but it partially reversed the increase in superoxide dismutase activity in older rats. OVX increased DNA damage in hippocampus of adult and older rats. VIT D per se reduced DNA damage, and when associated to OVX, it partially reversed this alteration. Additionally, OVX caused a telomere shortening in older rats, and VIT D was able to reverse such effect. Taken together, these results demonstrate that surgical menopause in rats causes hippocampal biochemical changes and VIT D appears, at least in part, to act in a beneficial way.

Methionine Administration in Pregnant Rats Causes Memory Deficit in the Offspring and Alters Ultrastructure in Brain Tissue

In the present work, we evaluated the effect of gestational hypermethioninemia on locomotor activity, anxiety, memory, and exploratory behavior of rat offspring through the following behavior tests: open field, object recognition, and inhibitory avoidance. Histological analysis was also done in the brain tissue of pups. Wistar female rats received methionine (2.68xa0μmol/g body weight) by subcutaneous injections during pregnancy. Control rats received saline. Histological analyses were made in brain tissue from 21 and 30xa0days of age pups. Another group was left to recover until the 30th day of life to perform behavior tests. Results from open field task showed that pups exposed to methionine during intrauterine development spent more time in the center of the arena. In the object recognition memory task, we observed that methionine administration during pregnancy reduced total exploration time of rat offspring during training session. The test session showed that methionine reduced the recognition index. Regarding to inhibitory avoidance task, the decrease in the step-down latency at 1 and 24xa0h after training demonstrated that maternal hypermethioninemia impaired short-term and long-term memories of rat offspring. Electron microscopy revealed alterations in the ultrastructure of neurons at 21 and 30xa0days of age. Our findings suggest that the cell morphological changes caused by maternal hypermethioninemia may be, at least partially, associated to the memory deficit of rat offspring.

Severe Hyperhomocysteinemia Decreases Creatine Kinase Activity and Causes Memory Impairment: Neuroprotective Role of Creatine

In the present study, we investigate the effect of severe hyperhomocysteinemia on biochemical (creatine kinase activity), behavioral (memory tests), and histological assessments (hippocampal volume). A possible neuroprotective role of creatine on hyperhomocysteinemia effects was also evaluated. Severe hyperhomocysteinemia was induced in neonate rats (starting at 6xa0days of age) by treatment with homocysteine (0.3–0.6xa0μmol/g body weight) for 23xa0days. Creatine (50xa0mg/kg body weight) was administered concomitantly with homocysteine. Controls received saline in the same volumes. Twelve hours after the last injection, the rats were submitted to behavioral tests [(recognition task (NOR)] and inhibitory avoidance (IA)]. Following behavioral assessment, the animals were perfused and decapitated, the brain removed for subsequent morphological analysis of the hippocampus. Another group of animals was used to test creatine kinase activity in hippocampus. The results showed that rats treated with homocysteine decreased (44%) the exploration of the novel object in NOR. In the IA task, homocysteine-treated animals presented decreased latencies to step down the platform in short- (32%) and long-term (18%) testings (3xa0h and 7xa0days, respectively), evidencing aversive memory impairment. Hippocampal volume was not altered by homocysteine administration. Hyperhomocysteinemia decreased (45%) creatine kinase activity, and creatine was able to prevent such effect probably by creatine kinase/phosphocreatine/creatine homeostasis, which serves as energy circuit within of the cell. This finding may be associated, at least in part, with memory improvement, suggesting that creatine might represent an effective adjuvant to protect against the effects of high homocysteine plasma levels.

Effects of previous physical exercise to chronic stress on long-term aversive memory and oxidative stress in amygdala and hippocampus of rats

Since stressful situations are considered risk factors for the development of depression and there are few studies evaluating prevention therapies for this disease, in the present study we evaluated the effect of previous physical exercise in animals subjected to chronic variable stress (CVS), an animal model of depression, on behavior tasks. We also investigated some parameters of oxidative stress and Na+, K+‐ATPase activity, immunocontent and gene expression of alpha subunits in amygdala and hippocampus of rats. Young male rats were randomized into four study groups (control, exercised, stressed, exercised + stressed). The animals were subjected to controlled exercise treadmill for 20 min,three times a week, for two months prior to submission to the CVS (40 days). Results show that CVS impaired performance in inhibitory avoidance at 24 h and 7 days after training session. CVS induced oxidative stress, increasing reactive species, lipoperoxidation and protein damage, and decreasing the activity of antioxidant enzymes. The activity of Na+, K+‐ATPase was decreased, but the immunocontents and gene expression of catalytic subunits were not altered. The previous physical exercise was able to improve performance in inhibitory avoidance at 24 h after training; additionally, exercise prevented oxidative damage, but was unable to reverse completely the changes observed on the enzymatic activities. Our findings suggest that physical exercise during the developmental period may protect against aversive memory impairment and brain oxidative damage caused by chronic stress exposure later in life.

Vitamin D partially reverses the increase in p-NF-κB/p65 immunocontent and interleukin-6 levels, but not in acetylcholinesterase activity in hippocampus of adult female ovariectomized rats

The aim of this study was to verify the effects of ovariectomy (OVX) and/or vitamin D supplementation (VIT D) on inflammatory and cholinergic parameters in hippocampus, as well as on serum estradiol and VIT D levels of rats. Ninety‐day‐old female Wistar rats were randomly divided into four groups: SHAM, OVX, VIT D or OVXu2009+u2009VIT D. Thirty days after OVX, VIT D (500 IU/kg/day) was supplemented by gavage, for 30 days. Approximately 12u2009h after the last VIT D administration, rats were euthanized and hippocampus and serum were obtained for further analyses. Results showed that OVX rats presented a decrease in estradiol levels when compared to control (SHAM). There was an increase in VIT D levels in the groups submitted to VIT D supplementation. OVX increased the immunocontent of nuclear p‐NF‐κB/p65, TNF‐α and IL‐6 levels. VIT D partially reversed the increase in p‐NF‐κB/p65 immunocontent and IL‐6 levels. Regarding cholinergic system, OVX caused an increase in acetylcholinesterase activity without changing acetylcholinesterase and choline acetyltransferase immunocontents. VIT D did not reverse the increase in acetylcholinesterase activity caused by OVX. These results demonstrate that OVX alters inflammatory and cholinergic parameters and that VIT D supplementation, at the dose used, partially reversed the increase in immunocontent of p‐NF‐Kb/p65 and IL‐6 levels, but it was not able to reverse other parameters studied. Our findings may help in the understanding of the brain changes that occurs in post menopause period and open perspectives for futures research involving VIT D therapies.

Gestational folic acid supplementation does not affects the maternal behavior and the early development of rats submitted to neonatal hypoxia-ischemia but the high supplementation impairs the dam’s memory and the Na+, K+ - ATPase activity in the pup’s hippocampus

Folic acid (FA) is a B‐complex vitamin important to the development of the fetus, being supplemented during pregnancy. Our recent findings showed that gestation supplementation (normal and excess doses) prevented the cognitive deficits and BDNF imbalance in adult rats that were submitted to neonatal hypoxia‐ischemia (HI). To better understand this protective effect, the present study aimed to evaluate whether FA supplementation could be related to (1) maternal behavior, memory and Na+, K+ ‐ ATPase activity in the hippocampus of the dams; (2) on somatic growth, early neurobehavioral development and Na+, K+ ‐ ATPase activity in the hippocampus of the offspring; and (3) the effects of this supplementation in pups submitted to neonatal HI. Pregnant Wistar rats were divided into three groups, according to the diet they received during gestation: standard diet (SD), supplemented with 2u2009mg/kg of FA (FA2 – normal dose) and supplemented with 20u2009mg/kg of FA (FA20 –excessive dose). At the 7th PND pups were submitted to the Levine‐Vannucci model of HI. During weaning the maternal behavior, the somatic growth and the neurobehavior development of pups were assessed. After weaning, the memory of the dams (by the Ox‐maze task) and the Na+, K+ ‐ ATPase activity in the hippocampus of both dams and offspring were evaluated. Considering the dams (1), both doses of FA did not alter the maternal behavior or the Na+, K+ ‐ ATPase activity in the hippocampus, but a memory deficit was observed in the high FA‐supplemented mothers. Considering the offspring (2), both FA doses did not affect the somatic growth or the neurobehavior development, but the FA20 pups had a decreased Na+, K+ ‐ ATPase activity in the hippocampus. The FA supplementation did not change the parameters evaluated in the HI rats (3) and did not prevent the decreased Na+, K+ ‐ ATPase activity in the hippocampus of the HI pups. These results indicate that normal FA supplementation dose does not influence the maternal behavior and memory and does not impact on the offspring early development in rats. Further studies are needed to confirm the effects of the high FA supplementation dose in the dams’ memory and in the Na+, K+ ‐ ATPase activity in the hippocampus of the offspring.

Forced Treadmill Exercise Prevents Spatial Memory Deficits in Aged Rats Probably Through the Activation of Na+, K+-ATPase in the Hippocampus

Regular physical activity has shown to improve the quality of life and to prevent age-related memory deficits. Memory processing requires proper regulation of several enzymes such as sodium–potassium adenosine triphosphatase (Na+, K+-ATPase) and acetylcholinesterase (AChE), which have a pivotal role in neuronal transmission. The present study investigated the effects of a treadmill running protocol in young (3 months), mature (6 months) and aged (22 months) Wistar rats, on: (a) cognitive function, as assessed in the Water maze spatial tasks; (b) Na+, K+-ATPase and AChE activities in the hippocampus following cognitive training alone or treadmill running combined with cognitive training. Animals of all ages were assigned to naïve (with no behavioral or exercise training), sedentary (non-exercised, with cognitive training) and exercised (20xa0min of daily running sessions, 3 times per week for 4xa0weeks and with cognitive training) groups. Cognition was assessed by reference and working memory tasks run in the Morris Water maze; 24 h after last session of behavioral testing, hippocampi were collected for biochemical analysis. Results demonstrated that: (a) a moderate treadmill running exercise prevented spatial learning and memory deficits in aged rats; (b) training in the Water maze increased both Na+, K+-ATPase and AChE activities in the hippocampus of mature and aged rats; (c) aged exercised rats displayed an even further increase of Na+, K+-ATPase activity in the hippocampus, (d) enzyme activity correlated with memory performance in aged rats. It is suggested that exercise prevents spatial memory deficits in aged rats probably through the activation of Na+, K+-ATPase in the hippocampus.