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.

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.

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.

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.

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.

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.

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.

Effects of duodenal-jejunal bypass on structure of diaphragm in western diet obese rats.

PURPOSE:: To evaluate the effects of duodenal-jejunal bypass (DJB) on the diaphragm muscle of obese rats fed on a western diet (WD) . METHODS:: Eighteen male Wistar rats were fed a standard rodent chow diet (CTL group) or WD ad libitum. After 10 weeks, WD rats were submitted to sham (WD SHAM) or duodenal-jejunal bypass (WD DJB). The structure, ultrastructure, collagen content and the morphometry of the neuromuscular junctions (NMJs) were analyzed two months after surgery. RESULTS:: WD SHAM rats displayed an increase in body weight, the Lee index and retroperitoneal and peri-epididymal fat pads compared to the CTL group. DJB did not alter these parameters. The muscle fiber structure and NMJs were similar in the WD SHAM and CTL groups. However, the WD SHAM group showed alterations in the fiber ultrastructure, such as loosely arranged myofibrils and Z line disorganization. In addition, WD SHAM animals presented a considerable amount of lipid droplets and a reduction in the percentage of collagen compared to the CTL group. DJB did not affect the structure or ultrastructure of the muscle fibers or the NMJs in the diaphragm of the WD DJB animals. CONCLUSION:: Duodenal-jejunal bypass did not improve the alterations observed in the diaphragm of western diet obese-rats.

Effects of duodenal-jejunal bypass on structure of diaphragm in western diet obese rats

Abstract Purpose: To evaluate the effects of duodenal-jejunal bypass (DJB) on the diaphragm muscle of obese rats fed on a western diet (WD) . Methods: Eighteen male Wistar rats were fed a standard rodent chow diet (CTL group) or WD ad libitum. After 10 weeks, WD rats were submitted to sham (WD SHAM) or duodenal-jejunal bypass (WD DJB). The structure, ultrastructure, collagen content and the morphometry of the neuromuscular junctions (NMJs) were analyzed two months after surgery. Results: WD SHAM rats displayed an increase in body weight, the Lee index and retroperitoneal and peri-epididymal fat pads compared to the CTL group. DJB did not alter these parameters. The muscle fiber structure and NMJs were similar in the WD SHAM and CTL groups. However, the WD SHAM group showed alterations in the fiber ultrastructure, such as loosely arranged myofibrils and Z line disorganization. In addition, WD SHAM animals presented a considerable amount of lipid droplets and a reduction in the percentage of collagen compared to the CTL group. DJB did not affect the structure or ultrastructure of the muscle fibers or the NMJs in the diaphragm of the WD DJB animals. Conclusion: Duodenal-jejunal bypass did not improve the alterations observed in the diaphragm of western diet obese-rats.

Maternal protein restriction during pregnancy and lactation affects the development of muscle fibers and neuromuscular junctions in rats.

INTRODUCTION: A balanced maternal diet is a determining factor in normal fetal development. The objective of this study was to evaluate the effects of maternal protein restriction during pregnancy and lactation on muscle fiber and neuromuscular junction (NMJ) morphology of rat offspring at 21 days of age. METHODS: Wistar rats were divided into a control group (CG), offspring of mothers fed a normal protein diet (17%), and a restricted group (RG), offspring of mothers fed a low-protein diet (6%). After a period of lactation, the animals were euthanized, and soleus muscles were obtained from pups for analysis. RESULTS: The soleus muscles of the RG exhibited an increase of 133% in the number of fibers and of 79% in the amount of nuclei. Moreover, the number of NMJs was lower in the restricted group than in the CG. CONCLUSIONS: Maternal protein restriction alters the normal development of the neuromuscular system. Muscle Nerve 55: 109-115, 2017.

Environmental enrichment attenuates the blood brain barrier dysfunction induced by the neonatal hypoxia-ischemia.

Environmental enrichment (EE) is considered an efficient neuroprotector against neonatal hypoxia-ischemia (HI). Nevertheless, the mechanisms involved are not yet clear. In this context, the aim of this study was to investigate the effects of neonatal HI and environmental stimulation in the hippocampus of rats at 3 different time points (PND 8, 22 and 60), evaluating some aspects of BBB structure and function. Seven-day-old Wistar rats were divided into four groups: a control group maintained in a standard environment (CTSE), a control group maintained in an enrichment environment (CTEE), an HI group maintained in a standard environment (HISE) and an HI group maintained in an enrichment environment (HIEE). At the 7th postnatal day (PND), rats were submitted to the Levine-Rice model of neonatal HI. This method consists of permanent occlusion of the right common carotid artery with subsequent exposure to hypoxia. Rats from CTEE and HIEE were stimulated with environmental enrichment. The EE protocol started 24h after HI, in which pup rats with their dams were stimulated in a maintained EE (PND 8-22). Subsequently, animals were submitted to daily EE (1h/day, PND 23-60). The expression of some proteins involved in BBB structure (ß-catenin, occludin, connexin-43, aquaporin-4, glut-1 and GFAP) were quantified by western blotting in the hippocampi of rats in three periods, at PND 8, 22 and 60. The BBB permeability and integrity was assessed by Evans blue staining and the immunohistochemistry for GFAP in the CA1 region of the hippocampus were also performed. The results showed an HI-induced decreased occludin expression at PND 22 and low levels of occludin, ß-catenin and GFAP at PND 60 in the hippocampus of the hypoxic-ischemic rats. Interestingly, in young and adult rats, EE reversed these effects. Evans blue extravasation into the brain parenchyma confirmed the BBB dysfunction brought on by HI. No differences were observed at PND 8, probably due to the immaturity of the BBB at this age. The present study makes an important contribution to understanding the mechanism of the hypoxic-ischemic brain damage and also to presents, for the first time, the recovery of BBB dysfunction as a possible pathway for the protective effect of EE.

Effects of aging and maternal protein restriction on the muscle fibers morphology and neuromuscular junctions of rats after nutritional recovery.

Changes in the nutritional status of mothers may predispose their offspring to neuromuscular disorders in the long term. This study evaluated the effects of maternal protein restriction during pregnancy and lactation on the muscle fibers and neuromuscular junctions (NMJs) of the soleus muscle in the offspring of rats at 365 days of age that had undergone nutritional recovery. Wistar rats were divided into two groups: control (CG)--the offspring of mothers fed a normal protein diet (17%) and restricted (RG)--offspring of mothers fed a low protein diet (6%). After lactation, the male pups received standard chow ad libitum. At 365 days, samples of soleus muscle were collected for muscle fiber analysis (HE staining, NADH-TR reaction and ultrastructure), intramuscular collagen quantification (picrosirius red staining) and NMJs analysis (non-specific esterase technique). The cross-sectional area of type I fibers was reduced by 20% and type IIa fibers by 5% while type IIb fibers increased by 5% in the RG compared to the CG. The percentage of intramuscular collagen was 19% lower in the RG. Disorganization of the myofibrils and Z line was observed, with the presence of clusters of mitochondria in both groups. Regarding the NMJs, in the RG there was a reduction of 10% in the area and 17% in the small diameter and an increase of 7% in the large diameter. The results indicate that the effects of maternal protein restriction on muscle fibers and NMJs seem to be long-lasting and irreversible.