Study On The Effect And Mechanisms Of Early Vitamin A Nutrition On Learning And Memory Function In Rat

Vitamin A is a necessary micronutrition in the whole life. Vitamin A and its derivates, the retinoids, play vital roles in the neurodevelopment and maintenance of the mature central nervous system, which exerts an essential role in both development and maintenance of the adult brain homeostasis. Marginal vitamin A deficiency is far more prevalent than severe deficiency in pregnant women and preschool children, but receives little attention due to no observable defects. However, postnatal intervention, rather than prenatal supplementation, is recommended for the vitamin A deficient cases by many countries to avoid the teratogenic effect of excess vitamin A during pregnancy.Adequate nutrition intake in early lifetime is related to the growth and development and the disease in adult time, and the center nervous system (CNS) is more susceptive to the early malnutrition. Adequate nutrition intake is very important to the neurodevelopment and maintenance of the CNS, which is directly affected by the nutrition state in CNS.Many maladies including attention deficit/hyperactivity disorder (ADHD), autistic spectrum disorders, and Alzheimer disease, all are believed to be the outcome of some abnormal process that unfolded as the brain was developing in utero or in the young child. Bioinformatics and clinical research all suggested that vitamin A nutrition is related to the neurodegenerative diseases, such as Alzheimer disease. We should pay more attention to the long-term effect of early malnutrition on the cognitive function, including protein, fat, mineral nutrition (Fe, Zn, Iodine, Cu and Se), Vitamins (A, C, D, E, and folic acid) and some long-chain undersaturation fatty acid. Recently, evidence has emerged that retinols are required for several aspects of adult brain function, especially for learning and memory function. It was reported that a diet completely free of vitamin A in adult rats could cause severe deficits in spatial learning and memory. Moreover, vitamin A supplementation can relieve the age-related cognitive impairment. However, little study reported the long-term effect of early vitamin A nutrition on the learning and memory and the relationship of this effect and neurodegenerative disease. Our previous population-based research reported the positive relationship between the serum retinoid level in newborn and the development of motor performance at two age-old. In the animal model, we also found marginal vitamin A deficiency (MVAD) began with pregnant time decreased the performance record in shuttle-box experiment and long-term potentiation (LTP) in CA1 area of hippocampus, and impaired the learning and memory of offspring rats. Moreover, different recover results were reported between the vitamin A supplementation at prenatal and postnatal period. But postnatal vitamin A supplementation is recommended in many countries, including china, for the neurotoxicity effect of vitamin A on the early developing CNS. So it is important to investigate the long-term effect of early vitamin A nutrition on the learning and memory function of offspring rats and"the critical window time"of the postnatal vitamin A supplementation.RA, an important active metabolite of vitamin A, regulates numerous aspects of neural development, including neuronal differentiation, neurite outgrowth, and the patterning of the anteroposterior axis of the neural tube. More recently, evidence has emerged that RA signaling pathway is required for several aspects of adult brain function as well, especially for learning and memory. Rats and mice fed a vitamin A deficient diet made more errors than controls in the radial maze spatial learning task. Replenishing dietary vitamin A reversed these deficits in rats and attenuated them in mice. Our previous study also reported atRA addition in the rat hippocampus slice can normalize the decreased LTP. Moreover, we also found there was no significant difference in the serum retinol concentration between the prenatal and postnatal vitamin A supplementation group, but was in LTP in CA1 area of rat hippocampus slice. These results suggested that RA signaling may play an important role in the irreversible cognitive impairment of offspring rats. There are several points at which the hippocampus response to RA could be regulated: 1. the uptake of retinol from the blood; the presence of the enzymes to convert retinol to RA; the presence of the RA nuclear receptor. RA translated protein, RA metabolism enzymes and receptors are all detected in the embryonic and matured brain. And the RA synthesis enzymes mainly expressed in the blood vessels and meanings surrounding the hippocampus. Moreover, it is likely that brain tissue synthesized RA more efficiently than other target tissues; the RA concentration is richer in hippocampus than other brain regions. So in the present study, we investigated the mechanism of that early vitamin A nutrition impairing the rat learning and memory function.Therefore, we found the MVAD pregnant and offspring rat model, and from 3 weeks before coitus, one group of rats was fed with a vitamin A adequate diet (6500 IU retinol/kg diet). Meanwhile, the other group was fed with a marginal vitamin A deficient diet (400 IU retinol/kg diet). These pup rats were divided into seven groups: (1)Control, rats pups that were vitamin A adequate began from gestational d 0 (G0), including gestation, lactation, and post-weaning (G0 to Postnatal 8 weeks); (2) MVAD (marginal vitamin A deficient group), rat pups that were marginal vitamin A deficient in gestation, lactation, and post-weaning (G0 to Postnatal 8 weeks); (3) VAS (vitamin A supplementation group ), rat pups that were vitamin A deficient in gestation followed by vitamin A adequacy to postnatal 8 weeks, with vitamin A supplementation (50000IU/kg weight/d, the administration of oils by gavage) from P0 to Postnatal 6 days. (4) OControl, rats pups that were vitamin A adequate began from gestational d 0 (G0), including gestation, lactation, and post-weaning (G0 to Postnatal 44 weeks); (5) OMVAD (old marginal vitamin A deficient group), rat pups that were marginal vitamin A deficient in gestation, lactation, and post-weaning (G0 to Postnatal 44 weeks); (6) OVAS1 (old vitamin A supplementation group 2), rat pups that were vitamin A deficient in gestation followed by vitamin A adequacy to postnatal 44 weeks, with vitamin A supplementation (50000IU/kg weight/d, the administration of oils by gavage) from P0 to Postnatal 6 days. (7) OVAS2 (old vitamin A supplementation group 2 ), rat pups that were vitamin A deficient in gestation followed by vitamin A adequacy to postnatal 44 weeks, with vitamin A supplementation (50000IU/kg weight/d, the administration of oils by gavage) for 7 days from Postnatal 4 weeks. In the experiment, we investigated the long-term effect of early MVAD and supplementation on the learning and memory behavior test by morris water maze; the brain protein concentration by biochemical analyzed method, and observed the ultramicrostructure deficit in hippocampus by electron microscope; the expression ofβ-amyloid (Aβ) protein and amyloid protein precursor (APP) in rat hippocampus by immunochemical method to study the effect of early MVAD on the Alzheimer disease morbidity. Second, in order to explore the effect of early vitamin A deficiency and supplementation on RA metabolism enzymes, we detected the mRNA expression of RA metabolism enzymes during the hippocampus development in early lifetime by real-time PCR, the retinol and RA concentration in hippocampus by HPLC. Third, we also detected the changes in the expression and subcellular localization of RARαin the rat hippocampus during postnatal development and the effect of early MVAD on this programming expression pattern by the real-time PCR, immunofluorescence and confocal laser-scanning microscope.The main results and conclusions were summarized as follows:1. Long-term cognitive function impairment was found in the early marginal vitamin A deficient offspring rats. MVAD group rats showed the learning and memory deficit in water maze test at post 8 and 44 weeks, which suggested the effect of MVAD on the cognitive function of offspring rats continued into aged stage. Different recover results were also founded between the vitamin A supplementation at birth and postnatal 4 weeks. In the vitamin A supplementation group at birth, the performance score in behavior test was significant lower than the control group at 8 weeks. However, there was no significant difference between these two groups at 44 weeks. These results may be related to the delay of cognitive function mature in the MVAD offspring rats. But in the vitamin A supplementation group at postnatal 4 weeks, the performance score in behavior test was significant lower than the control group both at 8 and 44 weeks, which suggested there is"the critical window time"for vitamin A supplementation in the early lifetime. The impairment of cognitive function can not be recovered by vitamin A supplementation out of this window time.2. As indicated by the ultrastructure of the hippocampus, persistent marginal vitamin A deficiency (MVAD) caused the damage of neurons and gliocytes in the hippocampus of offspring rats, increased accumulation of the lipofuscin in neurons, and aging of the nervous system. Immunohistochemical staining of the hippocampal slices by specific antibodies showed no obvious expression of Aβprotein in the hippocampus of neither normal 44-week old offspring rats nor MVAD ones; but the expression of APP protein was increased in the MVAD rats, which may be due to that MVAD interferes with the metabolism of APP and therefore leads to the accumulation of APP in the hippocampus. The increased expression of APP probably increases the risk of amyloid plaque. These data suggest that MVAD influences the long-term structure and function of the hippocampus, and may increase the risk of Alzheimer's disease, a degenerative neurological disorder, in the senectitude.3. The retinol-retinoic acid system in the circulation and hippocampus of offspring rats differentially responded to early MVAD. In the MVAD rats, the concentration of serum retinol was markedly decreased due to the reduced supply of food-derived vitamin A, but recovered to the normal level after the supplement of vitamin A. The concentration of retinol and retinoic acid in the hippocampus, however, did not alter, and instead, maintained within a stable range, responding to the same treatments. The correlation analysis indicated that neither the retinol nor retinoic acid in the hippocampus was apparently correlated with the change of learning and memory. So, the concentration of retinol and retinoic acid in the hippocampus could stand within a certain range, even if the supply of vitamin A or the concentration of serum retinol changed. This phenomenon may suggest a self-protection effect in the hippocampus. Altogether, the early marginal deficiency or supplement of vitamin A has little effect on the concentration of retinoic acid in the hippocampus. There maybe exist other mechanisms, but not the concentration of retinoic acid in the hippocampus, underlying the impairment of learning and memory induced by MVAD.4. The different responds of the RA system to early marginal vitamin A deficiency were different between in serum and in hippocampus tissue. This study observed the significant decreased in serum retinol concentration due to the vitamin A intake decreased, which can be recovered by vitamin A supplementation. Different from the change of retinol concentration in serum, hippocampal retinol and RA concentration was stable in the various vitamin A nutrition states. There was no significant relationship between the performance score in behavior test and retinol (or RA) concentration in hippocampus. So the hippocampal retinol and RA concentration can keep stable under the condition that vitamin A food intake and serum retinol concentration both changed, which may be resulted of the self-protection in hippocampus. And there may be other mechanisms rather than stable RA concentration in hippocampus resulted in learning and memory deficits in offspring rats.5. Our results showed that hippocampal RARαmRNA expression at P28 and P56 decreased by about 30% from P1 levels, but there was no difference between expression at P28 and P56. These results mean that RARαtranscript levels decreased during earlier postnatal development and then reached a stable level after weaning. As for RARαprotein expression, we obtained similar results. The peak rate of decrease appeared around the 4th postnatal week and was especially obvious in pyramidal cells in the hippocampus. RARαprotein levels were stable during later postnatal development. In pyramidal cells of areas CA1-CA3, the number of neurons expressing RARαduring later postnatal development was significantly reduced from earlier postnatal development. In granular layer cells, there was almost no RARαexpression observed whatsoever in areas CA1-CA3 during later postnatal development. Although RARαprotein was constantly expressed in DG granule cells, its expression level also gradually decreased. These results suggest that RARαreceptor expression plays an important role in earlier postnatal development and cognitive function of the hippocampus. 6. In this experiment, we also found the very interesting phenomenon that RARαreceptor protein in the hippocampus had different spatial and temporal expression patterns during postnatal hippocampal development. During earlier postnatal development, RARαexpression is mainly nuclear. Later, expression in cytoplasm gradually increases so there is both nuclear and cytoplasmic expression. During later postnatal development, RARαexpression is mainly cytoplasmic and can also be seen in some axons. It was found that RARαprotein translocated from the nucleus in earlier stages to the cytoplasm in later stages in the hippocampus, especially in pyramidal cells. These results indicate that RARαmay participated in the translation and play different roles during different stages of hippocampal development.7. Our results showed that early MVAD decreased the mRNA expression of RARαreceptor in hippocampus of pup rats at birth and postnatal 8 weeks. Moreover, the decreased RARαprotein expression and advanced cytoplasm dominant RARαprotein expression was also detected in hippocampus cell. These results suggested that early MVAD impaired the expression pattern of the RARαreceptor in postnatal hippocampus development, which may be one of the mechanisms account for the irreversible cognitive impairment reduced by miss"the critical window time"of vitamin A supplementation in early lifetime.