Mixed Exposure To Low Dose Heavy Metals Damaged Learning And Memory And The Mechanism Of Synaptic Plasticity In Rats

Objective:Human exposure to toxic heavy metals is a global challenge.Lead(Pb),Cadmiμm(Cd),and Mercury(Hg)have a lasting effects on the brain at low level.These heavy metals do not exist alone,and are always distributed in the environment where we live.This study established a low-dose of Pb,Cd,and Hg mixed exposure in rats models to explore the effect of low-dose heavy metal mixed exposure on learning and memory,and the related mechanisms of synaptic structural plasticity in the hippocampus,and provide the basis for further study of low-dose Pb,Cd,Hg mixed exposure to neurotoxicity and its mechanisms.Methods:1.Animals8 weeks old mature SD rats were mated with female and male ratios of 2:1,the pregnant SD rats were randomly divided into four groups and treated by drinking deionized water,1×MM group mixed venom(containing 25.00mg/L lead acetate trihydrate,7.50mg/L cadmiμm chloride and 0.15mg/L mercuric chloride of mixed venom),5×MM and 10×MM(equivalent to 1×MM group mixed venom 5-fold concentrations and 10-fold concentrations of the venom).After the birth of the pups,male pups were selected as experimental subjects and exposed to mixed venom through breast milk until weaning.At the Postnatal day 21(PND21),male pups were isolated and treated by drinking deionized water,1×MM group mixed venom(containing 8.33 mg/L lead acetate trihydrate,1.00mg/L cadmiμm chloride and 0.05mg/L mercuric chloride of mixed venom),5×MM and 10×MM(equivalent to 1×MM group mixed venom 5-fold concentrations and 10-fold concentrations of the venom)to poison the offspring of rats.The weight of rats was continuously monitored during the period of exposure until adulthood.2.Determination of Pb,Cd and Hg in blood and brain tissuesThe contents of Pb and Cd in rat blood and brain tissue were detected by inductively coupled plasma mass spectrometry(ICP-MS),and the Hg content in rat blood and brain tissues was measured by cold vapor atomic fluorescence spectrometry(CV-AFS).3.Rat learning and memory testsEvaluating spatial learning and memory ability in rats by using Eight-arm maze and Morris water maze tests.4.Dendritic spine structure detection in ratsDendritic spine density and type in DG,CA3,CA1 of hippocampus and prefrontal cortex were detected by Golgi-cox staining.5.Synaptic structure changes in ratsSynaptic density,PSD thickness,synaptic space,length of synaptic activation zone,nμmber of presynaptic membrane vesicles and curvature of synaptic interface changes in the CA1 region of the hippocampus were detected by transmission electron microscopy.6.Detection of ARP3 and N-WASp mRNA and protein expression levelsThe expression levels of ARP3 and N-WASp mRNA in hippocampus was detected by RT-PCR,and the protein expression levels of ARP3 and N-WASp in DG,CA3 and CA1 regions of hippocampus was observed by immunofluorescence mixed with laser scanning confocal microscope.Results:1.Low dose Pb,Cd and Hg mixed exposure on the growth and development of ratsIn PND0-PND51,the weight gain of rats decreased with the increase of exposure dose,and the difference was statistically significant(P<0.05).However,PND58-PND83 showed no significant difference in body weight among groups(P>0.05).2.The content of Pb,Cd and Hg in the blood and brain tissue of ratsThe contents of Pb,Cd and Hg in blood and brain tissue were detected by ICP-MS and CV-AFS.The results showed that the content of heavy metal in the blood of 1×MM rats: Pb 32.24±5.42μg/L、Cd 0.55±0.17μg/L、Hg 2.22±0.61μg/L,and the level of three heavy metals in the blood of the general population of our country(Pb 34.90μg/L、Cd 0.49μg/L、Hg 1.81μg/L).In addition,the MM levels in blood and brain tissue of rats increased in a dose-dependent manner(P<0.05).3.The effect of low dose Pb,Cd and Hg mixed exposure on learning and cognitive behavior in rats.The eight-arm maze and Morris water maze tests showed that the average latency time required by the rats in each dose group to find prey and the average escape latency time required to find the platform in each dose group were increased in a dose-dependent manner,and the 5×MM group and 10×MM group were longer than those in the blank control group,the difference was statistically significant(P<0.05).4.The effect of low dose Pb,Cd and Hg mixed exposure on the structure of dendritic spine in rats.Low dose Pb,Cd and Hg mixed exposure could cause a dose-dependent decrease in the density of dendritic spines in the hippocampus DG,CA3,CA1 and prefrontal cortex neurons,and all the exposed groups were lower than those in the blank control group(P<0.05).At the same time,the proportion of mushroom spines also decreased in a dose-dependent manner,and the three regions of CA3,CA1 and prefrontal cortex in the 5×MM group and 10×MM group were lower than those of the control group(P<0.05),and in the 1×MM group,only the CA1 area was lower than the control group,and the difference was statistically significant(P<0.05).The proportion of longthin spines in CA3 area of each dose group was higher than that in the control group,the difference was statistically significant(P<0.05).5.The effect of low dose Pb,Cd and Hg mixed exposure on synaptic structure in rats.Low dose Pb,Cd and Hg mixed exposure could cause the synaptic density,PSD thickness and the length of synaptic active band in the hippocampus CA1 region neurons decreased in a dose-dependent manner.The synaptic density,PSD thickness and the synaptic active band length of the hippocampal CA1 area neurons in the 5×MM group and the 10×MM group were lower than those of the control group,and the difference was statistically significant(P<0.05).The synaptic gap was only 10 × MM group is wider than the control group,the difference was statistically significant(P<0.05).6.The effect of low dose Pb,Cd and Hg mixed exposure on ARP3 and N-WASp mRNA and protein expression levels in hippocampus of rats.Low dose Pb,Cd and Hg mixed exposure could induce the expression of ARP3 and N-WASp protein in hippocampus DG,CA3 and CA1 decreased in a dose-dependent manner.The expression level of two protein in DG region was lower in the 5×MM group and 10×MM group than that in the control group(P<0.05).The expression level of two protein in CA3 area in MM groups was lower than that in the control group(P<0.05).The expression of N-WASp in the CA1 region in the 5 x MM group and 10×MM group was lower than that in control group,while ARP3 only 10×MM group was lower than the control group(P<0.05).The expression of ARP3 and N-WASp mRNA in hippocampus of each exposure group was not statistically different(P>0.05).Conclusion:1.This study established a rat model of learning and memory impairment induced by mixed exposure of low doses of Pb,Cd,and Hg.It was observed that the combination of low-dose Pb,Cd,and Hg exposures had a dose-dependent impairment in learning and memory.2.Golgi-cox staining and transmission electron microscopy were used to observe that the mixed exposure of low-dose Pb,Cd,and Hg can cause abnormal changes in synaptic structural plasticity in the rat hippocampus and prefrontal cortex.It shows the decrease of dendritic spines and synapses and the abnormal morphological structure in rats.3.Through immunofluorescence and confocal laser confocal technique,we found that the effect of low dose Pb,Cd and Hg mixed exposure on synaptic plasticity in rats may be related to the downregulation of N-WASp and ARP3 protein expression.