Effects Of Prenatal Low Level Lead Exposure On Learning And Memory In Offspring Rats And The Possible Mechanism

Lead, the most common environmental daily life pollutant, is one of the oldest of established environmental poisons. Lead can enter into the human's body and contributes to the damages of functions of many organs and systems. The central nervous system(CNS), especially, is the most important target of lead-mediated toxicity.The adverse effects from high level lead exposure have been decreased with the deeply knowledgement about lead toxicity and the taking of various preventive measures. But the low level lead exposure from environment and daily life is quite common. Because of the developing immature of blood brain barrier, the children are more sensitive than that of the adult to lead induced insults. So even low level lead exposure can give rise to functional disorder in children. The damage from low level lead exposure to children, especially to CNS and the mechanism of lead actions, is the hot point in public health research areas.Learning and memory, the basic functions of CNS, is one of the most important functions of CNS. As the key structure for learning and memory, hippocampus is the sensitive target of lead toxicity. It is approved that lead have very strong developmental neurotoxicity, even low level lead exposure can damage the developmental CNS. It is the critical period for development of CNS in fetus and infant. If prenatal lead exposure occurred, lead can be transported from mother to fetus through placenta easily and it also can come into the CNS of fetus through the incomplete blood brain barrier and do harm to the developmental CNS. As a result, the offspring's ability of learning and memory would be affected.Lead exposure during the gestation period is most responserble for the damage to the developing central nervous system of fetus caused by lead through various ways into the chhildrens'body. Previous studies indicate that impairment of later cognitive and neurobehavioral function result from lead exposure during early developing period(gestation/lactation) could persist long till to adult after the exposure has ceased. The developmental central nervous system (CNS) in organogenetic period and postnatal period are in a state of rapid growth, and more sensitive to toxic metals such as lead. Pb can make more serious damages to fetus and infant brains in prenatal stage than other stages. Despite intensive research, no consensus on the adverse effects and mechanisms of prenatal low level lead exposure to CNS in young offspring has resulted.In the present study we detected the Pb concentration in blood and content in the hippocampus in offspring rats after low level lead exposure during pregnancy. The changes of learning and memory in offspring rats were examined by Morris water maze test, shuttling and avoiding dark box test, and Y-type maze test. This study attempts to reveal the possible molecular mechanism of Pb induced neural development toxicity and provide the base of aristogenesis as well as prevent to the deficient of learning and memory of children.Part 1 The effects of prenatal low level lead exposure on the content of lead in blood and hippocampus of offspring rats.Objective: To establish the animal model of low level lead exposure during pregnancy, and to detect the changes of Pb concentration in blood and hippocampus in offspring rats and analyze the possible relationship between the Pb exposure and the deficient of learning and memory of offspring rats. This study in part provided the base data for revealing the possible mechanism of Pb to reduce the ability of learing and memory.Methods: The pregnant Wistar rats are housed separately and randomly divided into 4 groups and provided with doubly distilled water in the control group and 125,250,500mg/L lead acetate solution via drinking water in three exposured groups respectively during the pregnancy. The content of lead in blood and hippocampus of 21-day and 60-day old offsprings were measured by flow-injection hydride generation-atomic absorption spectrometry. At 1-day old offspring, the hippocampus was instead by whole brain.Results: The blood and hippocampus lead concentrations of 1-day old and 21-day old offsprings in the three lead exposed groups were significantly increased compared with the control group (P<0.05, P<0.01), and there was a good dose-response relationship between blood/hippocampus lead concentrations and the maternal exposure dosage. The concentration of Pb in blood and hippocampus of 60-day old offspring was no significantly difference in the lead exposed group compared with control group(P>0.05)。This result suggested that the concentration of Pb in blood and hippocampus of offspring rats increased following the increase of the maternal exposure dosage during pregnancy period.Conclusion: Prenatal exposure to low level lead make the lead concentrations of blood and hippocampus of the offspring significantly increased compared with the control group, and the increasing changes could continue to weaning at least. When the pups were mature, the concentration of Pb in blood and hippocampus decreased to normal levels.Part 2 The effects of prenatal low level lead exposure on learning and memory of offspring rats.Objective: To explore the effects prenatal low level lead exposure on learning and memory of offspring rats, and analyze the relationship between the impairments of learning /memory and lead concentrations in hippocampus. In this part, we want to observe the toxic effects of lead to CNS of offsprings rats.Methods: The learning and memory ability of the offspring rats were tested by the Morris water maze, Y-type maze, shuttling and avoiding dark box respedtivelty.Results: In Morris water maze test, the time to find the platform for 21-day old and 60-day old offspring in the three lead exposed groups significantly increased compared with the control group during the 4 days training (P<0.05, P<0.01). In the shuttling and avoiding dark box test, the times of initiative avoiding(TOIA) for 21-day and 60-day old offspring in the three lead exposed groups were significantly decreased, while the latency of the initiative avoiding(LOIA) and latency of the passive avoiding(LOPA) were significantly increased compared with the control group (P<0.05, P<0.01). In Y-type maze test, the ability of learning(A) and memory(B) and the memory retaining rate(C) for 21-day and 60-day old offspring in the three lead exposed groups were significantly decreased compared with control group(P<0.05, P<0.01).In this part we could found that the higher the Pb contents in hippocampus, the worse the ability of learning and memory of offspring rats. Especially, the deficits of learning and memory of soospring at mature stage(60-day old) in trearment group were still lower than that of control, while the Pb contents in hippocampus had decreased to a normal level. This results indicated the effect of lead to learning and memory could ontinue to the maturation period, and the recovery of the learning and memory might be need a long periods.Conclusion: Prenantal exposure to low level lead impaired the learning and memory ability of the offspring of the rats, and this kind of influence will continue till the offsprings'maturity. The deficits degrees of learning and memory were corelated with Pb contents in hippocampus of offspring rats. Part 3 The mechanism of prenatal low level lead exposure to learning and memory in young offspringObjective: To explore the effects of prenatal low level lead exposure to cell apoptosis, glial cells and neuron development of hippocampus of offspring rats. We are interested in the role of apoptosis as well as whether some neuronal specific genes, such as that encoding glial fibrillary acidic protein (GFAP), growth associated protein (GAP-43) and nerve growth factor (NGF), have changes in transcription and/or transcription level in rats after low levels Pb exposure.Methods: PI labeled apoptosis detection by flow cytometry and Terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling(TUNEL) were utilized to detect the hhippocampal cell apoptosis. The GFAP, GAP-43 and NGF protein expression in hippocampus were observed by immunohistochemistry and western blot, respectively. The GFAP, GAP-43 and NGF mRNA expression in hippocampus were observed by in situ hybridization and real-time PCR.Results: (1) The effects of prenatal low levels lead exposure on apoptosis of hippocampal cell in offspring rats.The apoptosis rates in hippocampus for 1-day old, 21-day old and 60-day old offspring examined by flow cytometry in each treatment group were significantly higher than that of control respectively(P<0.05, P<0.01), and there was a good dose-response relationship between apoptosis rates and the Pb concentration in hippocampus. The apoptosis index by TUNEL for 1-day old offspring in each treatment groups were significantly higher than that of control(P<0.05, P<0.01). For 21-day old and 60-day old offspring, the apoptosis index in medium and high dosage treatment groups were significantly higher than that of control group(P<0.05, or P<0.01), and showed a good dose-response relationship. There were positive correlation between apoptosis of hippocampus in offsprings and the deficits of learning and memory.(2) The effects of prenatal low levels lead exposure on astrocyte of hippocampus in offspring rats.Immunohistochemistry and Western blot showed that the protein expression of GFAP(include positive cell number, IOD and relative gray value) in hippocampus of 1-day old and 21-day old offspring in each treatment group were significantly higher than that of control(P<0.05, P<0.01), and there was a good dose-response relationship. But there were no significant difference at 60-day old offsprings. In situ hybridization and real time PCR showed that mRNA expression of GFAP(include positive cell number, IOD and△CT ) in hippocampus of 1-day old and 21-day old offspring in each treatment group were significantly higher than that of control(P<0.05, P<0.01), but no significant difference at 60-day old offspringa. The result above indicated that the promotion of GFAP protein and mRNA expression followed by the deficits of learning and memory ability of offsprings.(3) The effects of prenatal low levels lead exposure on neuron development of hippocampus in offspring rats.Immunohistochemistry showed showed that the GAP-43 protein expression(include area density and IOD) in hippocampus of 1-day old offspring in each treatment group were significantly lower than that of control(P<0.05, P<0.01). At 21-day, the value of area density and IOD in medium and high dosage treatment groups were decreased in hippocampus compared with the control group (P<0.05, P<0.01), and there were no significant difference at 60-day old offsprings. There was a good negactive dose-response relationship between the GAP-43 protein expression and lead in hippocampus, respectively. Western blot showed that the GAP-43 protein expression in hippocampus of 1-day old and 21-day old offspring in treatment groups singnificantly decreased compared with control, respectively(P<0.05, P<0.01), and there were no significant difference at 60-day old offsprings. In situ hybridization showed that the GAP-43 mRNA expression(include area density and IOD) in hippocampus of 1-day old offspring in each treatment group were significantly lower than that of control(P<0.05, P<0.01). At 21-day, the value of area density and IOD in medium and high dosage treatment groups were decreased in hippocampus compared with the control group (P<0.05, P<0.01), and there was a good negective dose-response relationship respectively, and there were no significant difference at 60-day old offsprings. Real time PCR showed that the GAP-43 mRNA expression in hippocampus of 1-day old and 21-day old offspring in treatment groups singnificantly decreased compared with control, respectively(P<0.05, P<0.01), and there were no significant difference at 60-day old offsprings.Immunohistochemistry and Western blot showed that the NGF protein expression(include area density, IOD and relative gray value) in hippocampus of 1-day old offspring in each treatment group were significantly lower than that of control(P<0.05, P<0.01), and there was a good negactive dose-response relationship between the NGFprotein expression and lead in hippocampus, respectively. There weas no significant difference at 60-day old offsprings. In situ hybridization showed that the NGF mRNA expression(include area density and IOD) in hippocampus of 1-day old offspring in each treatment group were significantly lower than that of control(P<0.05, P<0.01). At 21-day, the value of area density and IOD in medium and high dosage treatment groups were decreased in hippocampus compared with the control group (P<0.05, P<0.01), and there was a good negective dose-response relationship respectively, and there were no significant difference at 60-day old offsprings. Real time PCR showed that the NGF mRNA expression in hippocampus of 1-day old and 21-day old offspring in treatment groups singnificantly decreased compared with control, respectively(P<0.05, P<0.01), and there were no significant difference at 60-day old offsprings. The result above indicated that the decrease of GAP-43,NGF protein and mRNA expression could reduce the learning and memory ability of offsprings.Conclusion: It demonstrates that Pb could induce apoptosis in hippocampus, and increase the GFAP mRNA and protein expression as well as decrease the neuron outgrowth associated protein and factor expression (GAP-43 and NGF). The results in this part possibly implement that the mechanism of deficits of learning and memory of offspring rats after low level lead exposure during pregnancy might be through the apoptosis induction and increased level of GFAP as well as decreased the GAP-43 and NGF expression.