Lenir Orlandi Pereira

Lateralized and sex-dependent behavioral and morphological effects of unilateral neonatal cerebral hypoxia-ischemia in the rat

Neonatal cerebral hypoxia-ischemia (HI) is an important cause of neurological deficits. The Levine-Rice model of unilateral HI is a useful experimental tool, but the resulting brain damage is mainly restricted to one hemisphere. Since the rat presents morphological and biochemical asymmetries between brain hemispheres, behavioral outcome from this model is probably dependent on which hemisphere is damaged. We here investigated the effects of sex and lesioned hemisphere on the outcome of open field, plus maze, inhibitory avoidance and water maze tasks in adult rats previously submitted to neonatal unilateral HI. Females were more active than males in some of studied parameters and males presented better spatial learning. Hypoxia-ischemia caused spatial deficits independently of sex or damaged hemisphere. Right-HI increased locomotion only in males and caused working memory in females and on aversive learning in both males and females. Morphological analysis showed that right-HI animals presented greater reduction of ipsilateral striatum area, with females being more affected. Interestingly, males showed greater hippocampal volume. These results show that task performance and cerebral damage extension are lateralized and sex-dependent, and that the right hemisphere, irrespective of sex, is more vulnerable to neonatal cerebral hypoxia-ischemia.

Effects of daily environmental enrichment on behavior and dendritic spine density in hippocampus following neonatal hypoxia-ischemia in the rat.

Hypoxia-ischemia (HI) is the main cause of mortality in the perinatal period and morbidity, in survivors, which is characterized by neurological disabilities. The immature brain is highly susceptible to hypoxic-ischemic insult and is responsive to environmental stimuli, such as environmental enrichment (EE). Previous results indicate that EE recovered memory deficits in adult rats without reversing hippocampal atrophy related to HI. The aim of this study was to investigate behavioral performance in the open field and rota-rod apparatuses, in object recognition and inhibitory avoidance tasks, as well as dendritic spine density in the hippocampus, in rats undergoing HI and exposed to EE. Seven-day old male rats were submitted to the HI procedure and divided into 4 groups: control maintained in standard environment (CTSE), controls submitted to EE (CTEE), HI in standard environment (HISE) and HI in EE (HIEE). Behavioral and morphological parameters were evaluated 9 weeks after the environmental stimulation. Results indicate impairment in the object recognition task after HI that was recovered by enrichment; however the aversive memory impairment in the inhibitory avoidance task shown by hypoxic-ischemic rats was independent of the environment condition. Hypoxic-ischemic groups showed more crossing responses during the first minute in the open field, when compared to controls, but no differences were found between experimental groups in the rota-rod test. Dendritic spine density in the CA1 subfield of the right hippocampus (ipsilateral to the artery occlusion) was decreased after the HI insult, and increased in enriched controls; interestingly enriched HI rats did not differ from CTSE. In conclusion, EE was effective in recovering declarative memory impairment in object recognition and preserved hippocampal dendritic spine density loss after neonatal HI injury.

Long-term effects of environmental stimulation following hypoxia–ischemia on the oxidative state and BDNF levels in rat hippocampus and frontal cortex

Environmental enrichment recovers memory deficits without affecting atrophy of the hippocampus adult rats submitted to neonatal hypoxia-ischemia (HI). The present study was designed to investigate whether the modulation of brain oxidative status and/or BDNF content, as assessed in adulthood, are involved with the functional neuroprotection caused by environmental enrichment in animals receiving neonatal HI. Male Wistar rats, in the 7th postnatal day, were submitted to the Levine-Rice model of neonatal hypoxia-ischemia, comprising permanent occlusion of the right common carotid artery and a 90 min period of hypoxia (8% O(2)-92% N(2)). Starting 2 weeks after the HI event, animals were stimulated by the enriched environment (1 h/day for 9 weeks). Rats were sacrificed approximately 24 h after the end of enrichment period and some oxidative stress parameters, specifically the free radical levels, macromolecules damage and superoxide dismutase activity, in hippocampus and frontal cortex samples were determined. BDNF levels were also measured in the same encephalic structures. Indexes of macromolecules damage, TBARS levels and total cellular thiols, as well as free radical levels were unchanged in both studied structures. An increased SOD activity in the right hippocampus of HI group maintained in standard environment was found, this effect was reversed in HI enriched group. Moreover, BDNF levels were increased only in the hippocampus of non-stimulated HI group. These results suggest that the environmental enrichment protocol bearing cognitive protection is not associated to increases in BDNF expression nor SOD activity in hippocampus of the rats, as assessed in adulthood, submitted to neonatal hypoxia-ischemia.

ACUTE HOMOCYSTEINE ADMINISTRATION IMPAIRS MEMORY CONSOLIDATION ON INHIBITORY AVOIDANCE TASK AND DECREASES HIPPOCAMPAL BRAIN-DERIVED NEUROTROPHIC FACTOR IMMUNOCONTENT: PREVENTION BY FOLIC ACID TREATMENT

In the present study, we first investigated the effect of single homocysteine administration on consolidation of short- and long-term memories of inhibitory avoidance task in Wistar rats. We also measured brain-derived neurotrophic factor levels in the hippocampus and parietal cortex of rats. The influence of pretreatment with folic acid on behavioral and biochemical effects elicited by homocysteine was also studied. Wistar rats were subjected to a folic acid or saline pretreatment from their 22(nd) to 28(th) day of life; 12 h later they were submitted to a single administration of homocysteine or saline. For motor activity and memory evaluation we performed open-field and inhibitory avoidance tasks. Hippocampus and parietal cortex were obtained for brain-derived neurotrophic factor immunocontent determination. Results showed that homocysteine impaired short- and long-term memories and reduced brain-derived neurotrophic factor levels in the hippocampus. Pretreatment with folic acid prevented both the memory deficit and the reduction in the brain-derived neurotrophic factor immunocontent induced by homocysteine injection. Further studies are required to determine the entire mechanism by which folic acid acts and its potential therapeutic use for memory impairment prevention in homocystinuric patients.

Neonatal hypoxia-ischemia induces attention-deficit hyperactivity disorder-like behavior in rats

Attention-deficit hyperactivity disorder (ADHD) may be caused by genetic or environmental factors. Among environmental factors, perinatal complications are related, such as neonatal hypoxia-ischemia (HI). Thus, the aim of this study was to investigate whether HI contributes to the development of characteristics related to ADHD in adult rats, and to correlate the behavioral results with brain damage volume. Male Wistar rats were divided into 2 groups: HI and control. The HI procedure consisted of a permanent occlusion of the right common carotid artery followed by a period of hypoxia (90 min; 8% O₂ and 92% N₂) on the 7th postnatal day. Two months later, animals were evaluated in the open field test during a single 5-min session, and in the 5-choice serial reaction time task (5-CSRTT), over 25 weeks. Our results demonstrated that animals submitted to HI manifest cognitive impairments in task acquisition, deficits in sustained attention, and increases in impulsivity and compulsivity in response to task manipulation in the 5-CSRTT. Locomotor activity observed in open field did not differ between groups. Moreover, brain volume loss in the total hemisphere, cerebral cortex, white matter, hippocampus, and striatum were observed in HI animals, especially on the side ipsilateral to the lesion. From these results, we can infer that neonatal HI is an environmental factor that could contribute to the development of behavioral characteristics observed in ADHD that are associated with general brain atrophy.

Early environmental enrichment affects neurobehavioral development and prevents brain damage in rats submitted to neonatal hypoxia-ischemia

Our previous results demonstrated improved cognition in adolescent rats housed in environmental enrichment (EE) that underwent neonatal hypoxia-ischemia (HI). The aim of this study was to investigate the effects of early EE on neurobehavioral development and brain damage in rats submitted to neonatal HI. Wistar rats were submitted to the HI procedure on the 7th postnatal day (PND) and housed in an enriched environment (8th-20th PND). The maturation of physical characteristics and the neurological reflexes were evaluated and the volume of striatum, corpus callosum and neocortex was measured. Data analysis demonstrated a clear effect of EE on neurobehavioral development; also, daily performance was improved in enriched rats on righting, negative geotaxis and cliff aversion reflex. HI caused a transient motor deficit on gait latency. Brain atrophy was found in HI animals and this damage was partially prevented by the EE. In conclusion, early EE stimulated neurobehavioral development in neonate rats and also protects the neocortex and the corpus callosum from atrophy following HI. These findings reinforce the potential of EE as a strategy for rehabilitation following neonatal HI and provide scientific support to the use of this therapeutic strategy in the treatment of neonatal brain injuries in humans.

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 24 h 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 (1 h/day, PND 23–60). The expression of some proteins involved in BBB structure (β‐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, β‐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.

Enriched rehabilitation promotes motor recovery in rats exposed to neonatal hypoxia-ischemia.

Despite continuous improvement in neonatology there is no clinically effective treatment for perinatal hypoxia ischemia (HI). Therefore, development of a new therapeutic intervention to minimize the resulting neurological consequences is urgently needed. The immature brain is highly responsive to environmental stimuli, such as environmental enrichment but a more effective paradigm is enriched rehabilitation (ER), which combines environmental enrichment with daily reach training. Another neurorestorative strategy to promote tissue repair and functional recovery is cyclosporine A (CsA). However, potential benefits of CsA after neonatal HI have yet to be investigated. The aim of this study was to investigate the effects of a combinational therapy of CsA and ER in attempts to promote cognitive and motor recovery in a rat model of perinatal hypoxic-ischemic injury. Seven-day old rats were submitted to the HI procedure and divided into 4 groups: CsA+Rehabilitation; CsA+NoRehabilitation; Vehicle+Rehabilitation; Vehicle+NoRehabilitation. Behavioural parameters were evaluated pre (experiment 1) and post 4 weeks of combinational therapy (experiment 2). Results of experiment 1 demonstrated reduced open field activity of HI animals and increased foot faults relative to shams in the ladder rung walking test. In experiment 2, we showed that ER facilitated acquisition of a staircase skilled-reaching task, increased number of zone crosses in open-field exploration and enhanced coordinated limb use during locomotion on the ladder rung task. There were no evident deficits in novel object recognition testing. Delayed administration of CsA, had no effect on functional recovery after neonatal HI. There was a significant reduction of cortical and hemispherical volume and hippocampal area, ipsilateral to arterial occlusion in HI animals; combinational therapy had no effect on these morphological measurements. In conclusion, the present study demonstrated that ER, but not CsA was the main contributor to enhanced recovery of motor ability after neonatal HI.

Environmental stimulation improves performance in the ox-maze task and recovers Na+,K+-ATPase activity in the hippocampus of hypoxic–ischemic rats

In animal models, environmental enrichment (EE) has been found to be an efficient treatment for alleviating the consequences of neonatal hypoxia-ischemia (HI). However the potential for this therapeutic strategy and the mechanisms involved are not yet clear. The aim of present study is to investigate behavioral performance in the ox-maze test and Na+,K+-ATPase, catalase (CAT) and glutathione peroxidase (GPx) activities in the hippocampus of rats that suffered neonatal HI and were stimulated in an enriched environment. Seven-day-old rats were submitted to the HI procedure and divided into four groups: control maintained in standard environment (CTSE), control submitted to EE (CTEE), HI in standard environment (HISE) and HI in EE (HIEE). Animals were stimulated with EE for 9 weeks (1 h/day for 6 days/week) and then behavioral and biochemical parameters were evaluated. Present results indicate learning and memory in the ox-maze task were impaired in HI rats and this effect was recovered after EE. Hypoxic-ischemic event did not alter the Na+,K+-ATPase activity in the right hippocampus (ipsilateral to arterial occlusion). However, on the contralateral hemisphere, HI caused a decrease in this enzyme activity that was recovered by EE. The activities of GPx and CAT were not changed by HI in any group evaluated. In conclusion, EE was effective in recovering learning and memory impairment in the ox-maze task and Na+,K+-ATPase activity in the hippocampus caused by HI. The present data provide further support for the therapeutic potential of environmental stimulation after neonatal HI in rats.

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.