| With the acceleration of the industrialization and modernization of community, the environmental lead (Pb) pollution is getting very serious in our country. And the present condition of childhood lead poisoning is seriously concerned. Lead is a known neurotoxicant and exposures in children constitute a major public health problem because of the documented effects on cognitive development. Hippocampus is an important brain region, which plays a key role in learning and memory processes. Long-term potentiation (LTP) is a cellular model of synaptic plasticity, which may be representative of learning and memory processes in mammalian brain. And the N-methyl-D-aspartate (NMDA) receptor on the synaptosome in hippocampus is one of the biological bases of LTP. NMDA receptors are a subclass of excitatory amino acid receptors which play an important role in brain development, learning and memory processes. NMDA receptor consists of two subunit families, the NMDAR1 (NR1) and NMDAR2 (NR2), and the NR2 subunit family consists of four closelyrelated genes, the NR2A, NR2B, NR2C and NR2D. The genes encoding the NMDA receptor have been cloned. The gene expression of NMDAR subunits differs neuroanatomically and temporally. The NR1, NR2A and NR2B subunits dominate in developing hippocampus in mammalian.The NMDAR in developing brain is an important target of lead. Lead is a potent noncompetitive antagonist of the NMDA receptor. In vitro electro-physiological studies showed that lead could inhibit the NMDA activated whole cell currents, and that lead could also inhibit the opening of the single gate of NMDAR. Immature neurons are more sensitive to lead poison than mature neurons. But it is not known clearly whether Pb impairs the expression level of mRNA of NMDAR subunits. And the relationship between the effects and the exposure level of lead also isn't known yet.In the present study, we established a series of developing rat models exposed to low level lead. In order to know the impairment of lead on the NMDAR genes, we examined mRNA expression levels of the subunit NR2A and NR2B with RT-PCR method.Materials and MethodsMaterials:SD rats were obtained from the Center for Experimental Animals of Medical College of Zhejiang University. Analysis of Pb concentrations in blood was performed by graphite furnaceatomic absorption spectrometry (type AA700, Perkin Elmer, USA), and the standardized samples were friendly provided by the Center for Disease Control, USA. Trizol, Taq polymerase and AMV RTase were purchased from Roche (USA), Perkin Elmer (USA) and Sangon (Shanghai) separately. The primers of NR2A, NR2B and (3-actin were synthesized by Sangon.Methods:1. Establishing of Animal ModelsFemale rats were randomly assigned to distilled water containing 0, 0.025%, 0.05% or 0.075% lead acetate (PbAc-3H2O). Treatment was started two weeks prior to mating. Pups were weaned at 21 days of age and fed the same diet as the dams. At each developmental time point (7, 14, 21, and 28 days age), pups were euthanized by decapitation and hippocampus were dissected on an ice-chilled plate, stored at -70 癈 until used for RT-PCR studies. Blood samples of pups for lead analysis were obtained by heart puncture, and the samples of dams were obtained by thigh venous puncture.2. RT-PCR studyThe RNA of hippocampus was abstracted with Trizol, mRNA was reverse transcripted into cDNA by OligodT and AMV Rtase. Then PCR was performed in the tube containing the primers of NR2A or NR2B together with (3-actin separately. PCR producers were determined on 2% gel electrophoresis with the PCR marker. DNA densities were determined on DNA scanning machine to represent the quantity of mRNA.3. Statistical analysisAll statistical comparisons were conducted using one way analysis of variance (ANOVA). All values are expressed as mean盨D.Results1. No differences in body weight of dams were measured between any two groups (P>0.05). The blood lead levels of Pb-exposed dams were higher than those of control group(P<0.0... |
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