Zika Virus affects neurobehavioral development, and causes oxidative stress associated to blood-brain barrier disruption in a rat model of congenital infection.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with several neurodevelopmental outcomes after in utero infection. Here, we studied a congenital ZIKV infection model with immunocompetent Wistar rats, able to predict disabilities and that could pave the way for proposing new effective therapies. We identified neurodevelopmental milestones disabilities in congenital ZIKV animals. Also, on 22nd postnatal day (PND), blood-brain barrier (BBB) proteins disturbances were detected in the hippocampus with immunocontent reduction of ß_Catenin, Occludin and Conexin-43. Besides, oxidative stress imbalance on hippocampus and cortex were identified, without neuronal reduction in these structures. In conclusion, even without pups' microcephaly-like phenotype, congenital ZIKV infection resulted in neurobehavioral dysfunction associated with BBB and oxidative stress disturbances in young rats. Therefore, our findings highlighted the multiple impact of the congenital ZIKV infection on the neurodevelopment, which reinforces the continuity of studies to understand the spectrum of this impairment and to provide support to future treatment development for patients affected by congenital ZIKV.

Acrobatic training prevents motor deficits and neuronal loss in male and female rats following chronic cerebral hypoperfusion.

BACKGROUND: Chronic cerebral hypoperfusion in vascular dementia leads to memory and motor deficits; Physical exercise improves these aspects and promotes neuroprotection. Sexual dimorphism may significantly influence both ischemic and exercise outcomes. AIMS: The aim of this study was to investigate the effects of 2VO (Two-Vessel occlusion) and the acrobatic training on motor function, functional performance, and tissue loss in male and female rats. METHODS: Male and female rats were randomly divided into 4 groups: sham acrobatic, sham sedentary, 2VO acrobatic and 2VO sedentary. After 45 days of 2VO surgery, the animals received 4 weeks of acrobatic training. At the end, open field, beam balance and horizontal ladder tests were performed. Brain samples were taken for histological and morphological evaluation. RESULTS: Spontaneous motor activity in the open field was not affected by 2VO, on the other hand, an impairment in forelimb placement was observed after 2VO and acrobatic training prevented errors and improved hindlimb placement. Neuronal loss was found in the motor cortex and striatum after 2VO, especially in females, which was prevented by acrobatic training. CONCLUSION: Mild motor damage was found in animals after 2VO when refined movement was evaluated, probably associated to neuronal death in the motor cortex and striatum. The acrobatic exercise showed a neuroprotective effect, promoting neuronal survival and attenuating the motor deficit.

Acrobatic training prevents learning impairments and astrocyte remodeling in the hippocampus of rats undergoing chronic cerebral hypoperfusion: sex-specific benefits.

Background: Chronic cerebral hypoperfusion (CCH) leads to memory and learning impairments associated with degeneration and gliosis in the hippocampus. Treatment with physical exercise carries different therapeutic benefits for each sex. We investigated the effects of acrobatic training on astrocyte remodeling in the CA1 and CA3 subfields of the hippocampus and spatial memory impairment in male and female rats at different stages of the two-vessel occlusion (2VO) model. Methods: Wistar rats were randomly allocated into four groups of males and females: 2VO acrobatic, 2VO sedentary, sham acrobatic, and sham sedentary. The acrobatic training was performed for 4 weeks prior to the 2VO procedure. Brain samples were collected for morphological and biochemical analysis at 3 and 7 days after 2VO. The dorsal hippocampi were removed and prepared for Western blot quantification of Akt, p-Akt, COX IV, cleaved caspase-3, PARP, and GFAP. GFAP immunofluorescence was performed on slices of the hippocampus to count astrocytes and apply the Sholl's circle technique. The Morris water maze was run after 45 days of 2VO. Results: Acutely, the trained female rats showed increased PARP expression, and the 2VO-trained rats of both sexes presented increased GFAP levels in Western blot. Training, mainly in males, induced an increase in the number of astrocytes in the CA1 subfield. The 2VO rats presented branched astrocytes, while acrobatic training prevented branching. However, the 2VO-induced spatial memory impairment was partially prevented by the acrobatic training. Conclusion: Acrobatic training restricted the astrocytic remodeling caused by 2VO in the CA1 and CA3 subfields of the hippocampus. The improvement in spatial memory was associated with more organized glial scarring in the trained rats and better cell viability observed in females.

Neuroinflammation, blood-brain barrier dysfunction, hippocampal atrophy and delayed neurodevelopment: Contributions for a rat model of congenital Zika syndrome.

The congenital Zika syndrome (CZS) has been characterized as a set of several brain changes, such as reduced brain volume and subcortical calcifications, in addition to cognitive deficits. Microcephaly is one of the possible complications found in newborns exposed to Zika virus (ZIKV) during pregnancy, although it is an impacting clinical sign. This study aimed to investigate the consequences of a model of congenital ZIKV infection by evaluating the histopathology, blood-brain barrier, and neuroinflammation in pup rats 24 h after birth, and neurodevelopment of the offspring. Pregnant rats were inoculated subcutaneously with ZIKV-BR at the dose 1 × 107 plaque-forming unit (PFU mL-1) of ZIKV isolated in Brazil (ZIKV-BR) on gestational day 18 (G18). A set of pups, 24 h after birth, was euthanized. The brain was collected and later evaluated for the histopathology of brain structures through histological analysis. Additionally, analyses of the blood-brain barrier were conducted using western blotting, and neuroinflammation was assessed using ELISA. Another set of animals was evaluated on postnatal days 3, 6, 9, and 12 for neurodevelopment by observing the developmental milestones. Our results revealed hippocampal atrophy in ZIKV animals, in addition to changes in the blood-brain barrier structure and pro-inflammatory cytokines expression increase. Regarding neurodevelopment, a delay in important reflexes during the neonatal period in ZIKV animals was observed. These findings advance the understanding of the pathophysiology of CZS and contribute to enhancing the rat model of CZS.

Resistance exercise was safe for the pregnancy and offspring's development and partially protected rats against early life stress-induced effects.

Promising evidence points to gestational physical exercise as the key to preventing various disorders that affect the offspring neurodevelopment, but there are no studies showing the impact of resistance exercise on offspring health. Thus, the aim of this study was to investigate whether resistance exercise during pregnancy is able to prevent or to alleviate the possible deleterious effects on offspring, caused by early life-stress (ELS). Pregnant rats performed resistance exercise throughout the gestational period:they climbed a sloping ladder with a weight attached to their tail, 3 times a week. Male and female pups, on the day of birth (P0), were divided into 4 experimental groups: 1) rats of sedentary mothers (SED group); 2) rats of exercised mothers (EXE group); 3) rats of sedentary mothers and submitted to maternal separation (ELS group) and 4) rats of exercised mothers and submitted to MS (EXE + ELS group). From P1 to P10, pups from groups 3 and 4 were separated from their mothers for 3 h/day. Maternal behavior was assessed. From P30, behavioral tests were performed and on P38 the animals were euthanized and prefrontal cortex samples were collected. Oxidative stress and tissue damage analysis by Nissl staining were performed. Our results demonstrate that male rats are more susceptible to ELS than females, showing impulsive and hyperactive behavior similar to that seen in children with ADHD. This behavior was attenuated by the gestational resistance exercise. Our results demonstrate, for the first time, that resistance exercise performed during pregnancy seems to be safe for the pregnancy and offspring's neurodevelopment and are effective in preventing ELS-induced damage only in male rats. Interestingly, resistance exercise during pregnancy improved maternal care and it is reasonable to propose that this finding may be related to the protective role on the animals neurodevelopment, observed in our study.

Folic acid prevents behavioral impairment and Na(+), K(+) -ATPase inhibition caused by neonatal hypoxia-ischemia.

Folic acid plays an important role in neuroplasticity and acts as a neuroprotective agent, as observed in experimental brain ischemia studies. The aim of this study was to investigate the effects of folic acid on locomotor activity, aversive memory and Na(+),K(+)-ATPase activity in the frontal cortex and striatum in animals subjected to neonatal hypoxia-ischemia (HI). Wistar rats of both sexes at postnatal day 7 underwent HI procedure and were treated with intraperitoneal injections of folic acid (0.011 µmol/g body weight) once a day, until the 30th postnatal day. Starting on the day after, behavioral assessment was run in the open field and in the inhibitory avoidance task. Animals were sacrificed by decapitation 24 h after testing and striatum and frontal cortex were dissected out for Na(+),K(+)-ATPase activity analysis. Results show anxiogenic effect in the open field and an impairment of aversive memory in the inhibitory avoidance test in HI rats; folic acid treatment prevented both behavioral effects. A decreased Na(+),K(+)-ATPase activity in striatum, both ipsilateral and contralateral to ischemia, was identified after HI; a total recovery was observed in animals treated with folic acid. A partial recovery of Na(+),K(+)-ATPase activity was yet seen in frontal cortex of HI animals receiving folic acid supplementation. Presented results support that folic acid treatment prevents memory deficit and anxiety-like behavior, as well as prevents Na(+),K(+)-ATPase inhibition in the striatum and frontal cortex caused by neonatal hypoxia-ischemia.

Neonatal hypoxia-ischemia induces dysregulated feeding patterns and ethanol consumption that are alleviated by methylphenidate administration in rats.

Impulsivity, as observed in patients diagnosed with Attention-deficit/hyperactivity disorder (ADHD), can induce dysregulated behaviors such as binge eating and drug addiction. We previously demonstrated that neonatal hypoxia-ischemia (HI) resulted in ADHD-like behaviors in rats and that methylphenidate (MPH) administration (the first therapeutic option for ADHD) reversed these deficits. Here, we aimed at investigating addictive-like behaviors, such as the reward-based feeding behavior (using the BioDAQ monitor) and ethanol consumption (using the IA2BC procedure) in adult animals subjected to neonatal HI and treated with or without MPH. Male Wistar rats were divided into four groups (n = 10-12/group): control saline (CTS), CTMPH, HI saline (HIS) and HIMPH. The HI procedure was conducted at postnatal day (PND) 7 and behavioral analyses between PND 60-90, in which MPH (2.5 mg/kg, i.p.) was administered 30 min prior to each behavioral evaluation (6 sessions in BioDAQ and 12 sessions in the IA2BC protocol). HI animals had a dysregulated feeding intake shortly after eating a small piece of the palatable diet, and MPH reversed this dysregulated pattern. However, when the palatable diet was freely available, MPH stimulated a higher intake of this diet in the first exposure day, and this effect was potentialized in HIMPH rats. Increased ethanol intake was observed in HI rats, and MPH administration alleviated this behavior; contrarily, MPH treatment in control rats induced an increase in ethanol consumption. The present findings give additional support to the relationship between neonatal HI and ADHD but the differential response to MPH in control or HI animals highlights the importance of avoiding indiscriminate use of MPH by healthy individuals.

High gestational folic acid supplementation prevents hypoxia-ischemia-induced caspase-3 augmenting without changing synapsin and H3 methylation levels in the rat hippocampus.

Perinatal asphyxia is a peripartum event that can cause permanent sequelae to the newborns, affecting the brain development. Recently, it has been demonstrated that epigenetics mechanisms play an important role in this injury and that folic acid (FA) supplementation during pregnancy can affect these epigenetics modifications as well as gene expression. We have identified both positive and negative effects of FA treatment in rats submitted to a model of neonatal hypoxia-ischemia (HI). Considering that FA supplementation is already used in pregnant women and that HI occurs in the peripartum period, this study was designated to evaluate how gestational FA supplementation and neonatal HI affect: apoptosis (caspase-3) and expression of synaptic proteins (synapsin and PSD-95) and the methylation of histone H3 lysine (K) 4 and 27 in the rat hippocampus. Pregnant Wistar rats were divided according to the diets: standard (SD), supplemented with 2 mg/kg of FA or with 20 mg/kg of FA. HI procedure was performed at the 7th PND. Protein expression and H3 methylation were evaluated at the 60th PND in the rats' hippocampus. Neonatal HI increased caspase-3 expression decreased synapsin expression and reduced H3K4me2, -me3 and H3K27me2, -me3 in the ipsilateral hippocampus. FA only prevented the augment in caspase-3 expression. In conclusion, neonatal HI caused lasting effects on caspase-3-mediated cell death (prevented by the FA) and synaptic proteins in the rats' hippocampus. This is the first study to show that histone modifications may contribute to these pathological findings in the hippocampus of HI animals.

Acrobatic exercise recovers object recognition memory impairment in hypoxic-ischemic rats.

Neonatal hypoxia-ischemia (HI) can lead to cognitive impairments and motor dysfunction. Acrobatic exercises (AE) were proposing as therapeutic option to manage HI motor deficits, however, the cognitive effects after this treatment are still poorly understood. Therefore, we evaluated the effects of AE protocol on memory impairments and brain plasticity markers after Rice-Vannucci HI rodent model. Wistar rats on the 7th postnatal day (PND) were submitted to HI model and after weaning (PND22) were trained for 5 weeks with AE protocol, then subsequently submitted to cognitive tests. Our results showed recovery in novel object recognition (NOR) memory, but not, spatial Morris Water Maze (WM) memory after AE treatment in HI rats. BDNF and synaptophysin neuroplasticity markers indicate plastic alterations in the hippocampus and striatum, with maintenance of synaptophysin despite the reduction of total volume tissue, besides, hippocampal HI-induced ipsilateral BDNF increased, and striatum contralateral BDNF decreased were noted. Nevertheless, the exercise promoted functional recovery and seems to be a promising strategy for HI treatment, however, future studies identifying neuroplastic pathway for this improvement are needed.

Effects of exercise modalities on BDNF and IL-1ß content in circulating total extracellular vesicles and particles obtained from aged rats.

There are evidences about the involvement of systemic factors, such as brain-derived neurotrophic factor (BDNF), on functional exercise effects. Although aerobic exercise can impact circulating extracellular vesicles and particles (EVPs) cargo, other exercise modalities were not studied. Taken that BDNF and anti-inflammatory effects have been related to functional outcomes, and BDNF and IL-1ß have been detected in circulating EVPs, our aim was to evaluate circulating total EVPs profile from adult and aged Wistar rats submitted to exercise modalities, namely aerobic, acrobatic, resistance or combined for 20 min, 3 times a week, during 12 weeks. A modality- and age-dependent effect on total EVPs cargo was observed; aerobic exercise induced an augment in BDNF and IL-1ß in EVPs from aged rats, while acrobatic and combined exercise modalities reduced IL-1ß content in EVPs from adult ones. Besides, all exercise modalities attenuated aging-induced CD63 changes in circulating total EVPs; this finding can be involved with reduced mortality rate and improved memory performance previously observed. Changes on EVPs profile, such as increased CD63 levels can be related, at least in part, to an exercise-induced healthier global status. Additionally, aerobic exercise-induced effects on BDNF and IL-1ß levels might indicate additional benefits in aged individuals.

Methylphenidate treatment increases hippocampal BDNF levels but does not improve memory deficits in hypoxic-ischemic rats.

BACKGROUND: Methylphenidate (MPH) is a stimulant drug mainly prescribed to treat cognitive impairments in attention-deficit/hyperactivity disorder (ADHD). We demonstrated that neonatal hypoxia-ischemia (HI) induced attentional deficits in rats and MPH administration reversed these deficits. However, MPH effects on memory deficits after the HI procedure have not been evaluated yet. AIMS: We aimed to analyze learning and memory performance of young hypoxic-ischemic rats after MPH administration and associate their performance with brain-derived neurotrophic factor (BDNF) levels in the prefrontal cortex and hippocampus. METHODS: Male Wistar rats were divided into four groups (n=11-13/group): control saline (CTS), control MPH (CTMPH), HI saline (HIS) and HIMPH. The HI procedure was conducted at post-natal day (PND) 7 and memory tasks between PND 30 and 45. MPH administration (2.5 mg/kg, i.p.) occurred 30 min prior to each behavioral session and daily, for 15 days, for the BDNF assay (n=5-7/group). RESULTS: As expected, hypoxic-ischemic animals demonstrated learning and memory deficits in the novel-object recognition (NOR) and Morris water maze (MWM) tasks. However, MPH treatment did not improve learning and memory deficits of these animals in the MWM-and even disrupted the animals' performance in the NOR task. Increased BDNF levels were found in the hippocampus of HIMPH animals, which seem to have been insufficient to improve memory deficits observed in this group. CONCLUSIONS: The MPH treatment was not able to improve memory deficits resulting from the HI procedure considering a dose of 2.5 mg/kg. Further studies investigating different MPH doses would be necessary to determine a dose-response relationship in this model.

Neonatal hypoxia-ischemia caused mild motor dysfunction, recovered by acrobatic training, without affecting morphological structures involved in motor control in rats.

The aim of this study was to evaluated motor function and morphological aspects of the components involved in motor control (sensorimotor cortex, spinal cord, sciatic nerve, neuromuscular junctions and skeletal muscle) in male Wistar rats exposed to a model of neonatal hypoxic-ischemic encephalopathy (HIE) and the possible influence of different physical exercise protocols - treadmill and acrobatic. Male Wistar rats at the 7th post-natal day (PND) were submitted to the HIE model and from the 22nd until 60th PND the exercise protocols (treadmill or acrobatic training) were running. After the training, the animals were evaluated in Open Field, Ladder Rung Walking and Rotarod tasks and after samples of the motor control components were collected. Our results evidenced that the acrobatic training reversed the hyperactivity and anxiety, caused locomotion improvement and decreased brain atrophy in HIE animals. We did not find morphological differences on sensorimotor cortex, spinal cord, sciatic nerve, neuromuscular junctions and skeletal muscle in the animals submitted to HIE model. These intriguing data support the statement of the Rice-Vannucci model does not seem to reproduce, in structures involved in control function, the damage found in humans that suffer HIE. Regarding the protocols of exercise, we proposed that the acrobatic exercise could be a good therapeutic option especially in children affected by neonatal HIE and can be responsible for good results in cognitive and motor aspects.

Methylphenidate administration reverts attentional inflexibility in adolescent rats submitted to a model of neonatal hypoxia-ischemia: Predictive validity for ADHD study.

Perinatal complications such as birth asphyxia were associated with a higher risk for Attention-Deficit/Hyperactivity Disorder (ADHD) in humans. Data from a rat model of neonatal hypoxia-ischemia (HI) have revealed inattention, impulsive behavior and dopamine (DA) disturbances in the prefrontal cortex (PFC), confirming the face validity and construct validity for ADHD study. However, the predictive validity (similar therapeutic efficacy of the pharmacological treatment available in the clinic) should be considered. Therefore, we aimed to investigate the effects of methylphenidate (MPH) - the treatment of choice for ADHD - on exploratory and attentional flexibility behaviors and DA-related proteins in the PFC of animals submitted to neonatal HI. Male Wistar rats were divided into four groups: control saline (CTS, n = 12), control MPH (CTMPH, n = 12), HI saline (HIS, n = 13) and HIMPH (n = 12). The HI procedure was conducted at postnatal day (PND) 7 and behavioral measures between PND 30-40, followed by protein analysis in the PFC. The MPH administration (2.5 mg/kg, i.p.) occurred 30 min prior each behavioral session and euthanasia for western blot analysis. We observed that the MPH increased the locomotor activity in the open field especially in HI rats. In the attentional-set shifting task, the MPH reversed the HI- induced attentional inflexibility, but impaired the task acquisition in control rats. Neonatal HI resulted in lower DA D2 receptors expression but also decreased DA transporter (responsible for DA reuptake) and increased pTH (phosphorylated-tyrosine hydroxylase) levels in the PFC, probably to compensate the dysfunctional DA transmission. This compensation was higher in the HIMPH group and it could explain the improvement in the attentional flexibility as well as the increased locomotor activity in this group. Taken this data together, we can assume the predictive validity of the HI model for the ADHD study concerning the impact of MPH treatment on attentional parameters.

Prefrontal Cortex Dopamine Transporter Gene Network Moderates the Effect of Perinatal Hypoxic-Ischemic Conditions on Cognitive Flexibility and Brain Gray Matter Density in Children.

BACKGROUND: Genetic polymorphisms of the dopamine transporter gene (DAT1) and perinatal complications associated with poor oxygenation are risk factors for attentional problems in childhood and may show interactive effects. METHODS: We created a novel expression-based polygenic risk score (ePRS) reflecting variations in the function of the DAT1 gene network (ePRS-DAT1) in the prefrontal cortex and explored the effects of its interaction with perinatal hypoxic-ischemic-associated conditions on cognitive flexibility and brain gray matter density in healthy children from two birth cohorts-MAVAN from Canada (n = 139 boys and girls) and GUSTO from Singapore (n = 312 boys and girls). RESULTS: A history of exposure to several perinatal hypoxic-ischemic-associated conditions was associated with impaired cognitive flexibility only in the high-ePRS group, suggesting that variation in the prefrontal cortex expression of genes involved in dopamine reuptake is associated with differences in this behavior. Interestingly, this result was observed in both ethnically distinct birth cohorts. Additionally, parallel independent component analysis (MAVAN cohort, n = 40 children) demonstrated relationships between single nucleotide polymorphism-based ePRS and gray matter density in areas involved in executive (cortical regions) and integrative (bilateral thalamus and putamen) functions, and these relationships differ in children from high and low exposure to hypoxic-ischemic-associated conditions. CONCLUSIONS: These findings reveal that the impact of conditions associated with hypoxia-ischemia on brain development and executive functions is moderated by genotypes associated with dopamine signaling in the prefrontal cortex. We discuss the potential impact of innovative genomic and environmental measures for the identification of children at high risk for impaired executive functions.

Systemic administration of a nitric oxide synthase inhibitor impairs fear sensitization in the plus-maze.

The aim of the present study was to evaluate the occurrence of fear sensitization in rats previously treated with an inhibitor of the NO syntheses and submitted to Trial1/Trial2 plus-maze (PM) procedure. Male Wistar rats received a systemic treatment with N(omega)-nitro-L-Arginine-methyl ester (L-NAME; 5, 10 or 50 mg kg(-1)) and were submitted to PM Trial1. In the following day the animals were re-exposed to the PM with no drug administration (Trial2). Some standards spatial-temporal measures, such as the percentage of entries (% Open arm entries) and time spent (% Open arm time) in the open arms and risk assessment frequency were recorded and used to estimate the animal level of fear sensitization in PM Trial2. The results showed that animals receiving L-NAME (50 mg kg(-1)) displayed increased % Open arm entries and % Open arm time in Trial2 in relation to the group receiving 0.9% saline, which is compatible with impaired fear/anxiety acquisition during Trial1/Trial2 PM procedure. In addition, rats treated with L-NAME (50 mg kg(-1)) exhibited low level of risk assessment in Trial2 in relation to the group treated with 0.9% saline, which indicates low level of fear/anxiety during PM re-exposure. The number of entries into the enclosed arms was not changed by any L-NAME treatment, which suggests no bias of the drug treatments on animal locomotor activity. The data suggest that NO synthesis may mediate the fear sensitization process in the PM.

Chronic early postnatal glutaric acid administration causes cognitive deficits in the water maze.

Glutaric acidemia type I (GA I) is an autosomal recessive metabolic disorder caused by glutaryl-CoA dehydrogenase deficiency leading to predominant accumulation of glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric acid (3HG) in body fluids and tissues. The clinical manifestations of GA I are predominantly neurological. Although the pathophysiological mechanisms responsible for the brain damage of this disease are virtually unknown, they are thought to be due to the neurotoxic actions of GA and 3HG. Therefore, in the present work we investigated whether chronic exposure of GA (5 micromol g of body weight(-1), twice per day), the major metabolite accumulating in GA I, during early development (from the 5th to the 28th day of life) could alter the cognitive performance of adult rats in the Morris water maze, open field and elevated plus maze tasks. Control rats were treated with saline in the same volumes. GA administration provoked an impairment of spatial performance in the water maze since adult rats pretreated with GA were not able to remember the previous location of the platform spending significantly less time in the training quadrant. In contrast, GA chronic administration did not affect rat performance in the open field and elevated plus maze tasks, indicating that motor activity and anxiety was not changed by GA. The results provide evidence that early chronic GA treatment induces long-lasting spatial behavioral deficit.

Early enriched housing results in partial recovery of memory deficits in female, but not in male, rats after neonatal hypoxia-ischemia.

Our previous results indicated that stimulation by daily environmental enrichment (EE) recovered memory deficits without affecting hippocampus damage in adult male rats submitted to neonatal hypoxia-ischemia (HI). The present study investigated whether early continuous housing in an enriched environment would be effective in preventing spatial and recognition memory both in adolescent and adult female and male rats, as well as the possible benefits of continuous EE in alleviating hippocampal and striatal atrophy consequent to the neonatal HI. Wistar rats in the 7th PND were submitted to the HI and, in the day after, were housed in an enriched environment (8th-30th PND). Subsequently, performance of animals in the novel-object recognition and in two water maze tasks was assessed; in adulthood, animals' behavior was reassessed in the water maze. Rats were sacrificed and both hippocampal volume and striatal area were estimated following the completion of behavioral study. Post-HI cognitive deficits in the object recognition test were completely recovered by the EE. However, memory impairment in the water maze was only partially prevented by EE; this effect was observed especially in female rats on the working memory protocol. As for the morphological assessment, there was no enrichment effect over the loss of hippocampus volume and striatum area. In conclusion, present data indicate that early housing in EE caused performance recovery in object recognition and a partial improvement in the working memory spatial task in adolescent females after neonatal HI; however no effects of enrichment were revealed in adult animal's performance or in the extension of tissue atrophy of hippocampus and striatum consequent to HI.

Prefrontal cortex dysfunction in hypoxic-ischaemic encephalopathy contributes to executive function impairments in rats: Potential contribution for attention-deficit/hyperactivity disorder.

OBJECTIVES: The attention-deficit/hyperactivity disorder (ADHD) compromises the quality of life of individuals including adaptation to the social environment. ADHD aetiology includes perinatal conditions such as hypoxic-ischaemic events; preclinical studies have demonstrated attentional deficits and impulsive-hyperactive outcomes after neonatal hypoxic and/or ischaemic intervention, but data are missing to understand this relationship. Thus, the aim of this study was to evaluate executive function (EF) and impulsivity, and tissue integrity and dopaminergic function in the prefrontal cortex (PFC) of rats submitted to hypoxia-ischaemia (HI). METHODS: At postnatal day (PND) 7, male Wistar rats were divided into control (n = 10) and HI groups (n = 11) and the HI procedure was conducted. At PND60, the animals were tested in the attentional set-shifting (ASS) task to EF and in the tolerance to delay of reward for assessment of impulsivity. After, morphological analysis and the dopaminergic system were evaluated in the PFC. RESULTS: Animals subjected to HI had impairments in EF evidenced by a behavioural inflexibility that was correlated to PFC atrophy. Moreover, HI animals presented reduced D2 receptors in the ipsilateral side of ischaemia in the PFC. CONCLUSIONS: Animals submitted to HI presented impaired EF associated with tissue atrophy and dopaminergic disturbance in the PFC.

Folic acid supplementation during pregnancy prevents cognitive impairments and BDNF imbalance in the hippocampus of the offspring after neonatal hypoxia-ischemia.

Folic acid (FA) supplementation (400 µg/day) has been recommended during pregnancy to prevent neural tube defects. However, in some countries, flours are required to be fortified with FA, possibly increasing the levels of this vitamin in pregnant women. Our previous studies have evidenced a dual effect of the FA treatment in a rat model of neonatal hypoxia-ischemia (HI). Aiming to better correlate with humans, this paper evaluated the effects of two different levels of FA supplementation during pregnancy on memory parameters and neuronal survival and plasticity in the hippocampus of rats submitted to the neonatal HI. During pregnancy, female Wistar rats received one of these diets: standard (SD), supplemented with 2 mg/kg of FA or with 20 mg/kg of FA. At the 7th PND, rats suffered the HI procedure. At the 60th PND rats were evaluated in the open field, Morris water maze, novel-object recognition and inhibitory avoidance tasks. Furthermore, neuronal density, synaptophysin densitometry and BDNF concentration were assessed in the hippocampus. Both doses of FA prevented the HI-induced memory impairments. The supplementation reversed the BDNF late increase in the hippocampus of the HI rats, but did not inhibit the neuronal death. In conclusion, FA supplementation during pregnancy prevented memory deficits and BDNF imbalance after neonatal HI. These findings are particularly relevant because neuroprotection was achieved even in the high level of FA supplementation during pregnancy, indicating that this intervention would be considered secure for the offspring development.

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 2 mg/kg of FA (FA2 - normal dose) and supplemented with 20 mg/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.