Folic Acid Prevents Behavioral Impairment and Na+,K+-ATPase Inhibition Caused by Neonatal Hypoxia–Ischemia
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- Carletti, J.V., Deniz, B.F., Miguel, P.M. et al. Neurochem Res (2012) 37: 1624. doi:10.1007/s11064-012-0757-6
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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.
KeywordsHypoxia–ischemiaFolic acidInhibitory avoidanceNa+,K+-ATPaseOpen-field test
Neonatal hypoxia–ischemia (HI) is a harmful event affecting structural and functional integrity of the nervous system . It occurs due to complications in the perinatal period and is associated with neurological disorders such as cerebral palsy, mental retardation, epilepsy, sensory motor impairments and learning disorders [2, 3].
The pathogenesis of neonatal hypoxia–ischemia is a complex process where the energy failure, followed by glutamatergic excitotoxicity, inflammation and oxidative stress are the main metabolic events . It is known that the immature brain of the newborn is particularly vulnerable to oxidative damage due to its limited antioxidant capacity . Following that, it has been shown that a decrease in the delivery of blood flow leads to the formation of free radicals that, in large amounts, have detrimental effects such as lipid peroxidation, as well as proteins, enzymes, carbohydrates and DNA insults . At the cellular level, the lack of oxygen supply causes a decrease of adenosine triphosphate (ATP) due to anaerobic glycolysis; such reduction in ATP concentration may impair the Na+,K+-ATPase activity, which is critical to the maintenance of cell membrane excitability and functions. Wyse et al.  have demonstrated that recovery of Na+,K+-ATPase activity in the hippocampus is responsible for neuroprotection induced by brain ischemic preconditioning. This finding indicates the important role of Na+,K+-ATPase activity to in cellular neuroprotection.
Hypoxia–ischemia usually causes damage to different brain structures such as prefrontal cortex, hippocampus, striatum and thalamus . Previous studies from our laboratory demonstrated hippocampal, striatal and cortical atrophy in rats submitted to neonatal HI event, as compared to control animals, associated with several cognitive disabilities such as spatial long-term memory in Morris water maze , object recognition memory  and aversive memory in inhibitory avoidance task .
Neuroprotective agents, like resveratrol, melatonin and vitamins, have been used to mitigate the damage caused by cerebral HI [4, 11], however no treatment has been fully efficient to counteract all consequences of neonatal HI. Folic acid (FA) is a water-soluble B vitamin, involved in homocysteine remethylation and nucleotide biosynthesis, and is associated with the prevention of neural-tube defects  and is used in treatment of megaloblastic anemia . It is well known that adequate availability of essential nutrients involved in the cellular metabolism of one-carbon is essential for development and maintenance of brain function and neuroplasticity . It has been found that folic acid supplementation prevents ischemic events, independently of homocysteine metabolism [15, 16], and exerts protective effects in Alzheimer’s patients . In addition, it was demonstrated that folic acid also plays an antioxidant role when used in experimental hyperhomocysteinemic rats, an effect associated with prevention of brain Na+,K+-ATPase activity inhibition .
Considering the available evidence that folic acid exerts therapeutic effects in experimental models of neurological disorders, the aim of this study was to investigate whether folic acid treatment would affect behavioral performance, as assessed in inhibitory avoidance and open field tasks, of rats subjected to neonatal HI. Its effect on Na+,K+-ATPase activity in frontal cortex and striatum, structures susceptible to HI damage and associated with sensory motor  and cognitive functions , was also studied.
Materials and Methods
The Levine rat model , as modified by Rice et al. , to neonatal rats was employed. At the seventh postnatal day (PND), animals were anesthetized with halothane and the right common carotid artery was identified through a longitudinal neck incision, isolated from the nerve and vein, and permanently occluded with surgical silk thread. Animals were then allowed to recover for 15 min under a heating lamp and returned to their dams. After 2 h, groups of five pups were placed in airtight jars and exposed to a humidified nitrogen–oxygen mixture (92 and 8 %, respectively) for 90 min with the chamber partially immersed in a 37 °C water bath to maintain a constant thermal environment. Controls were sham-operated, i. e., they were submitted to anesthesia and neck incision, but did not receive arterial occlusion or hypoxia.
Folic Acid Chronic Treatment
Folic acid solution (0.011 μmol folic acid/g body weight) was injected intraperitoneally, once a day, from the 7th to the 30th day-of-age [18, 23]. This dosing regimen was chosen according to studies of Matté and colleagues [18, 24], where protective effects of FA administration on damaged brain structures and cognitive function in young rats were observed. Control animals received saline solution in the same volumes as those administrated to folic acid-treated groups. Folic acid was obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Open field test was used to evaluate motor activity and anxiety. It was performed in a square wooden apparatus measuring 50 × 50 cm with 40 cm walls, with a frontal glass wall, divided into 12 equal quadrants according to Netto et al. . Twenty-four hours after the end of pharmacological treatment, animals were placed facing the left corner of the arena and the free exploration was observed during 5 min. The analyzed variables were latency to leave the start position, number of crossings among quadrants, number of rearings and number of fecal boli. The arena was thoroughly cleaned with a 20 % of ethanol solution between sessions.
Twenty-four hours after the open field test, animals were trained on step-down inhibitory avoidance task . A 50 cm wide, 25 cm high, 25 cm deep acrylic box was used. The left-most 7 cm of the box floor was occupied by a 3 cm high platform. The rest of the box floor was a grid of parallel stainless steel bars (15-mm-diameter) spaced 7 mm apart. Animals were gently placed onto the platform, facing the corner, and latencies to step down placing their four paws on the grid were measured with an automatic device. On stepping down, they received a 0.6 mA, 60 HZ scrambled footshock for 2–3 s, and were returned to their home cages. Animals were tested for retention 24 h later. Test session was procedurally similar to the training, except that the footshock was omitted; step-down latency in test session was used as an index of retention.
Na+,K+-ATPase Activity Assay
As for the biochemical assay, animals were sacrificed by decapitation approximately 24 h after the behavioral study, on the 34th postnatal day; right and left frontal cortex and striatum were dissected out and instantaneously placed in liquid nitrogen and stored at −70 °C until biochemical assays. Brain structures were homogenized in 10 volumes (1:10, w/v) of 0.32 M sucrose solution containing 5.0 mM HEPES and 0.1 mM EDTA, pH 7.4.
The reaction mixture for Na+,K+-ATPase activity assay contained 5.0 mM MgCl2, 80.0 mM NaCl, 20.0 mM KCl and 40.0 mM Tris-HCl, pH 7.4, in final volume of 200 mL. The reaction was initiated by ATP addition. Controls were carried out under the same conditions with the addition of 1.0 mM ouabain. Na+,K+-ATPase activity was calculated by the difference between the two assays, as described by Wyse et al. . Released inorganic phosphate (Pi) was measured by the method of Chan and colleagues . Specific enzyme activity was expressed as nmol Pi released per min per mg of protein. All samples were run in duplicate.
Two-way analysis of variance (ANOVA) was performed, with lesion and treatment as factors, followed by Duncan’s test to analyze data from open field task and Na+,K+-ATPase activity. Data from Inhibitory avoidance was analyzed using the Kruskall Wallis ANOVA followed by Mann–Whitney test when indicated. All statistical tests were performed using the Statistica® software package running on a compatible personal computer; differences were considered statistically significant whenever p < 0.05.
As regards to enzyme activity in the frontal cortex, ANOVA demonstrated a significant effect associated with lesion (right: F(1,20) = 69.9; p < 0.05; left: F(1,20) = 6.5; p < 0.05). Post hoc Duncan’s test indicated that HI-S group also had lower activity of Na+,K+-ATPase on both sides, when compared with the group CT-S. Additionally, hypoxic-ischemic group treated with folic acid did not differ of that CT groups, indicating once again a presence of a protective effect (Panel B of Fig. 3).
This study focused on the possible neuroprotective action of folic acid supplementation on the behavioral and biochemical effects caused by neonatal HI. It was shown that HI caused an anxiogenic response in the open field and a cognitive deficit in the inhibitory avoidance task, as well an inhibition of Na+,K+-ATPase activity both in striatum and frontal cortex; folic acid treatment prevented all these effects.
General motor activity and anxiety-like behaviors were assessed in the open-field. Here, data obtained indicated that animals submitted to hypoxic-ischemic event treated with saline displayed increased anxiety-like behavior, as seen in higher latency to leave the initial quadrant, and made less crossings, compared with the other groups. Folic acid administration prevented these effects in HI animals. It has been reported that folate deficiency before the birth of rats increased anxiety and decreased the number of rearings in the plus maze test . Corroborating this data, in humans, folic acid intake restriction was associated with depression in male smokers  and in older women .The present study analyzed males and females data together, because a previous study demonstrated that 30- and 45-day-old rats of both genders presented similar defecation, ambulation, and rearing scores in the open field test .
Inhibitory avoidance test measures learning through the animal’s latency to descend from the platform, which is motivated by the experience of an aversive event . Present data corroborate previous findings indicating that neonatal HI causes aversive memory impairment  as well as spatial memory deficits in adolescent animals [34, 35]. Moreover, the present study showed that animals subjected to HI and treated with FA had no cognitive impairment on the inhibitory avoidance test. This interesting result indicate, for the first time, that folic acid may be considered as a neuroprotective strategy for reversal or alleviate cognitive deficits caused by neonatal hypoxia–ischemia.
As regard to Na+,K+-ATPase activity assessment, in frontal cortex and striatum data analysis indicated that HI animals treated with saline had a decrease in Na+,K+-ATPase activity on both hemispheres (ipsilateral and contralateral), comparing with control group. These results are consistent with a recent report which identified a decrease in Na+,K+-ATPase activity in cortex 1 h after the hypoxic-ischemic insult . Additionally, present results demonstrated that FA supplementation was able to reverse decreased Na+,K+-ATPase activity, specially in striatum. We could suggest that this finding is partially responsible by the behavioral effects identified on inhibitory avoidance and open field tasks. This suggestion corroborates findings reported by Matté and colleagues  which state that hyperhomocysteinemic rats treated with FA had a recovery of decreased Na+,K+-ATPase activity and memory impairments. These authors suggested that these findings probably can be attributed to the antioxidant properties of folic acid [15, 18]. In the present study, it was not investigated oxidative parameters. However, we believe that this hypothesis is acceptable and is strongly supported by a recent study which demonstrated increased superoxide dismutase and catalase enzymes activity and decreased lipid peroxidation in cortex, midbrain and cerebellum regions in the old rats submitted to folic acid supplementation . Regarding to ischemic events, a prospective study correlates the increase in plasma folate with reduced risk for hemorrhagic stroke . Moreover Kremer and Grosso  studied the relationship of FA with hypoxic-ischemic events in the perinatal period. These authors observed that mutations in the gene encoding 5,10-methylenetetrahydrofolate reductase in women with a nutritional deficiency of folate, may represent a risk factor for hypoxic-ischemic encephalopathy in neonates. When analyzing Na+,K+-ATPase activity in the frontal cortex present data showed partial recovery of inhibition in the HI-FA group, being observed only on the contralateral side to lesion. It is reasonable consider that contralateral side to be less affected by HI than ipsilateral side, as previously stated .
In conclusion, here present data suggest that hypoxia–ischemia produces anxiogenic effects and aversive memory deficits, and that treatment with folic acid was able to prevent these effects. When evaluating the Na+,K+-ATPase activity, treatment with FA was able to reverse the inhibition of enzyme activity partially in striatum and totally in frontal cortex. Thus, folic acid may be considered a candidate for neuroprotection after a neonatal hypoxic-ischemic event and, probably, this effect is partially mediated by recovering of the Na+,K+-ATPase enzyme activity. Therefore, more studies are needed to clarify the mechanisms involved in the neuroprotective effect of folic acid.
This work was supported in part by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq—Brazil) and (Fundação de Amparo à Pesquisa do estado do Rio Grande do Sul (FAPERGS).