Study About The Effects And Mechanisms Of Early Vitamin A Deficiency On Children's Neurodevelopment And Synaptic Plasticity Signaling Pathway In Rat

PartⅠEffects of vitamin A nutrition during pregnancy on children's neurodevelopment at two years oldOBJECTIVE: To investigate the correlation of cord blood vitamin A level at delivery and the neurodevelopment at two years age, and evaluate the effect of vitamin A nutrition during pregnancy on neurodevelopment of offspring.METHODS: The present research was a prospective cohort study and the subjects were 158 children born to nonsmoking women who gave birth at four big hospitals in Tongliang, Chongqing, China, between March 2, 2005 and May 24, 2005. Maternal blood and umbilical cord blood were collected at delivery to detect the serum retinol concentration by high-performance liquid chromatography (HPLC). A questionnaire about demographic information as well as personal-social information was administered by trained interviewers after delivery. The neonatal birth weight, length and head circumference were also measured. Intellectual development was evaluated by Gesell Development Schedule (GDS) at two years old.RESULTS: A total of 158 maternal–neonate pairs were recruited and complete epidemiological and clinical data were obtained from 157 pairs. At two years of age, 122 children were tested with the GDS.①Serum retinol level of maternal was significantly higher than that of cord (p<0.0001), but without any correlativity between them (p>0.05). The prevalence of vitamin A deficiency (VAD) and marginal VAD (MVAD)/suspected subclinical VAD (SSVAD) in neonates were 60.1% and 35.1%, respectively. The prevalence of maternal VAD and MVAD/SSVAD were 12.8% and 43.9%, respectively.②The vitamin A placental transport rate (VA-PTR) was (0.72±0.33). When the maternal retinol levels varied from 0.18μmol/L to 2.56μmol/L, the neonatal retinol levels keep in the range of (0.30-1.40)μmol/L.③After adjusting for potential confounders, VA-PTR was positively associated with motor area development quotients (DQ) (p=0.0082) and average DQ (p=0.0053). Cord VA level was positively related with language area (p=0.0265) and social area DQ (p=0.0191).④The adaptive area and average DQs in high cord VA group was higher than those in low VA group (p<0.05).⑤After adjusting for potential confounders, cord VA level and VA-PTR were positively associated with birth head circumference and birth weight, respectively.CONCLUSIONS:①It is a moderate public health problem of pregnant VAD in the locality, which needs urgent measures to make it better.②Cord retinol level is significantly lower than maternal retinol level, while there is no correlation between them.③Placenta plays an important role in regulating VA transportation from mothers to fetus, which might be helpful to maintain the neonatal VA levels in a relatively small scope when the maternal VA levels varied in a large range.④Adequate vitamin A nutrition during pregnancy has beneficial influence on children's neurodevelopment. The effect of vitamin A status on motor development might be more obvious than adaptation, language and personal-social development.⑤There were relationships between birth weight or head circumference and cord retinol level or VA-PTR.PartⅡEffects of marginal vitamin A deficiency beginning from pregnancy on hippocampus synaptic plasticity signaling pathway related genes expression in young ratsOBJECTIVE: To investigate if the effect of MVAD from pregnancy on learning and memory in offspring rats was more serious than that of VAD from born, and the impact of VA supplementation from born. The effects of MVAD from pregnancy on hippocampus synaptic plasticity signaling pathway related genes expression in young ratsMETHODS: Thirty-two female rats were randomly divided into control, MVAD, vitamin A supplementation (VAS) and postnatal vitamin A deficiency (PVAD) groups in this study. In control group the dams and pups were fed with normal diet(VA 6500 IU/kg). In MVAD group the dams and pups were fed with MVAD diet(VA 400 IU/kg). In VAS and PVAD groups, the pups were exchanged at delivery. The offspring in VAS group were given oral vitamin A (50IU/g/d) from postnatal day 1 to day 7. Eight female pups were respectively killed postnatal day 1(P1d), P2w, P4w and P8w in control and MVAD groups. A ten days Morris water maze (MWM) test was conducted in the four groups at P7w. Eight female pups with MWM test or non-MWM test were killed in control group at P8w. We monitored the serum vitamin A concentration by HPLC. The mRNA expression of retinoic acid receptor RARα, RARβ, NMDA receptor subunit NR1, NR2A, NR2B, CAMKⅡα, Arc and CBP in hippocampus were detected by fluorescence quantitative PCR(FQ-PCR).RESULTS:①The dams'serum VA concentration in MVAD was lower than that of control, and they always did not have the manifestation of VAD. The serum VA concentration of MVAD group was lower than that of control group at P8w (P<0.05), and there was no difference between VAS and control groups. While the VA level in PVAD group was between control and VAS.②Morris water maze test: During the spatial acquisition test, rats in MVAD group showed longer escape latency than control, VAS and PVAD groups. The latency in PVAD group was between that in control and VAS groups. In the probe trial test, the percent time in target quadrant of MVAD and VAS group was significantly smaller than that of control group. There was no difference between PVAD and control group. In the spatial reversal test, the percent time in target quadrant of control and VAS group was significantly bigger than that of MVAD and PVAD groups at the third day. There was no difference between MVAD and PVAD groups.③There was no difference of the serum VA level between MWM and non-MWM pups, while the expression of RARα, NR1, NR2A, Arc, CAMKⅡαand CBP mRNA in hippocampus were higher in MWM pups than those in non-MWM pups (p<0.05), and the mRNA levels of RARβand NR2B remained unchanged after MWM test (p>0.05). There were positively correlation between the mRNA level of RARαand NR1, NR2A, NR2B, Arc and CBP.④FQ-PCR showed that there was an increase of the expression of NR1 in hippocampus from P1d to P2w, and then decrease gradually. The expression of NR2A and NR2B showed ascending trend and downtrend, respectively from P1d to P8w. The mRNA levels of NR1 and NR2B of control group was significantly higher in most time point than those in MVAD group. The ratio of NR2A to NR2B at P2w was lower in control than that in MVAD group (p<0.05).⑤The expression of Arc and CAMKⅡαincreased gradually from P1d to P8w. The expression of Arc mRNA in control group was significantly higher than that in MVAD group at P4w and P8w(p<0.05), and CAMKⅡαlevel in control group was higher than that in MVAD group at P2w and P4w(p<0.05), and there was no difference of CBP level between the two groups.CONCLUSIONS:①There are significantly effects of MVAD from embryonic and postnatal VAD on the spatial learning, short-term memory, long-term memory and the ability to learn new things at P7w. The effects are more serious in MVAD group than that in PVAD group. VA supplementation from born could not fully restore these abilities at P7w.②There are increase of some synaptic plasticity signaling pathway related genes after MWM test, and RARαmay have close relationship with there changes in the formation and maintain of learning and memory.③MVAD from pregnancy have influence on the early postnatal mRNA expression of important genes related to synaptic plasticity pathway, such as NR1, NR2B, Arc and CAMKⅡα, and could impaire the formation and maintain of long-term potentiation (LTP) in pups.Part III Marginal vitamin A deficiency from embryonic influences the development of hippocampus N-methyl-D-aspartate receptor subunit at critical period via retinoic acid receptor in ratsOBJECTIVE: To explore if MVAD from embryonic influenced the development of hippocampus N-methyl-D-aspartate receptor subunit NR1 at critical period via RARα.METHODS: Sixteen female rats were randomly divided into control and MVAD groups. The method of animal model was the same as partⅡ. Eight female pups in each group were respectively killed at P1d, P2w, P4w and P8w. We detected the expression and subcellular localization of RARα,RARβand NR1 in postnatal hippocampus by FQ-PCR, immunofluorescence, confocal laser scanning microscope and Western Blotting.RESULTS:①The expression of RARαmRNA gradually declined from P1d to P8w, while there was no significantly changes of RARβpostnatal. MVAD resulted the decreased levels of RARα, RARβand NR1 mRNA (P<0.05).②Hippocampal RARαprotein expression at P2w, P4w and P8w decreased compared with P1d (P<0.05), but there was no difference between expression at P4w and P8w. At P1d, abundant RARαprotein located at cytoplasm and neuritis, and then located at nucleus of neurons at P2w. Later, it shifted to the cytoplasm gradually, and expressed in the cytoplasm and some neuritis at P8w. We obtained similar results from Western blotting. No distinct expression of RARβwas detected by immunofluorescence at P1d and P4w in hippocampus. The western blotting showed that the expression of RARβincreased gradually postnatal, and located mainly in the nucleus. Immunofluorescence and western blotting results showed that the expression of NR1 declined with development and located in the cytoplasm.③MWM test resulted in the increase of RARαand NR1 protein in the cytoplasm.④Confocal laser scanning microscope displayed that a mass of RARαand NR1 co-located in the cytoplasm at P1d in hippocampus.⑤The fluorescence intensity and western blotting results showed that RARαand NR1 in MVAD group was lower than those in control group (p <0.05) and still located in the cytoplasm postnatal. There was no significantly difference of RARβbetween MVAD and control groups (p>0.05).CONCLUSIONS:①MVAD from embryonic could results in the decreased mRNA and protein expression of RARα, but with no obvious effect on those of RARβ.②The time and subcellular location-developmental programmed expression of RARαin postnatal hippocampus in rats may be related to the different roles it played during the formation of learning and memory. The cytoplasm location of RARαmay participated in the translation regulation.③MVAD from embryonic may influence the development of hippocampus NR1 at critical period via RARα, and this could be one of the mechanisms that how could MVAD from embryonic affect the synaptic plasticity signaling pathway, and result in impairment of learning and memory in offspring rats.