| Objective:Environmental heavy metal pollution has become a public health problem that seriously affects people's health,and the health hazards caused by the exposure of lead(Pb),cadmium(Cd),and mercury(Hg)are more serious,these heavy metals usually coexist in the living environment,and early exposure can cause children's cognitive dysfunction.It has been found that enriched environmental intervention can improve cognitive ability recently.So,can enriched environmental intervention improve the cognitive dificits induced by heavy metal co-exposure? And what is the possible molecular mechanism? In this study,Pb,Cd,and Hg mixed aqueous solutions were exposed in drinking water to establish rats models of early-life(pre-embryo to weaning)and continuous(embryos to adult)co-exposure of heavy metals and enriched environmental intervention rats model.The purpose was to explore the possible mechanism of the restoration of cognitive deficits induced by low-dose Pb,Cd,and Hg co-exposure in rats.And to provide a scientific basis for further study of the repair of neurotoxicity damage induced by heavy metal pollutants.Methods:1.The animal models9 weeks specific pathogen free(SPF)-grade SD rats were matched in cages by male rats: female rats =1:2,the female rats successfully conceived were randomly divided into the control group(only drinking deionized water),continuous exposure group(the exposure of male offsprings in embryos and weaning was performed by the mothers drinking a mixed aqueous solution containing 250 mg/L lead acetate trihydrate,75 mg/L cadmium chloride,and 1.5 mg/L mercuric chloride,after weaning,the offspring rats were fed a mixed aqueous solution containing 83.333 mg/L lead acetate trihydrate,10 mg/L cadmium chloride,and 0.5 mg/L mercuric chloride until adulthood),preweaning exposure group(the male offsprings' exposure in embryos and weaning was performed by the mothers drinking a mixed aqueous solution containing 250 mg/L lead acetate trihydrate,75 mg/L cadmium chloride,and 1.5 mg/L mercuric chloride,after weaning,the male offspring drink deionized water until adulthood),and enriched environmental intervention group(the male offsprings' exposure in embryos and weaning was performed by the mothers drinking the same concentration of Pb,Cd,Hg mixed aqueous solution with the preweaning exposure group,and deionized water was used after the weaning,on the other hand,the enriched environmental intervention was given from the first day after birth),and feeding rats to adulthood.2.Determination of heavy metals content in whole blood and brainWhole blood and brain were digested with microwave digestion.The contents of Pb and Cd in rat whole blood and brain were measured by inductively coupled plasma mass spectrometry(ICP-MS),and the content of Hg was detected by cold vapor atomic fluorescence spectrometry(CV-AFS).3.Neurobehavioral testingThe Morris water maze test was performed to examine the spatial learning and memory ability of rats.The radial arm maze test was performed to examine the working memory ability of rats.4.Detection of dendritic spines and synapses ultrastructureGolgi-Cox staining was used to observe the density and morphology of dendritic spine in hippocampal CA1,CA3,DG areas and prefrontal cortex.Transmission electron microscope was used to observe the density and morphology of synaptic ultrastructure in hippocampal CA1 area.5.Detection of Snk and SPAR mRNA and protein expressionThe mRNA expression of serum-inducible kinase(Snk)and Spine-associated Rap guanosinetriphosphatase activating protein(SPAR)in the hippocampus of rats was detected by reverse transcription PCR(RT-PCR).The expression of Snk and SPAR proteins in the hippocampus of rats was detected by immunofluorescence,confocal laser scanning and Western Blot.6.Detection of hippocampal neuronal damageTransmission electron microscopy was used to observe the damage of rat hippocampal neuronal cell and organelles,and the levels of reactive oxygen species(ROS)and apoptosis in rat hippocampal neurons were determined by flow cytometry.Results:1.Effects of enriched environment on the contents of heavy metals in whole blood and brain of rats exposed to Pb,Cd and Hg.ICP-MS and CV-AFS revealed that the contents of Pb,Cd and Hg in rats' whole blood and brain in continuous exposure group were higher than in control group(P<0.05).In addition,in the enriched environment group and preweaning exposed group,Pb was not detected in whole blood and brain,the contents of Cd and Hg were lower than in continuous exposure group(P<0.05),and higher than in control group(P<0.05).2.Effects of enriched environment on cognitive behaviors of rats co-exposed to Pb,Cd and Hg.Enriched environment improved the spatial learning and memory and working memory deficits in rats induced by co-exposed to Pb,Cd,and Hg.In the Morris water maze,it was found that the escape latency of the finding platform in continuous exposure group and preweaning group was significantly higher than in control group(P < 0.05),while the escape latency of the enriched environment intervention group was significantly lower than in preweaning group(P < 0.01),and the difference was not statistically significant compared with the control group(P > 0.05).In the radial arm maze,the latency of rats looking for food in continuous exposure group and preweaning exposure group was significantly higher than in control group(P<0.01),while the latency of the rats in the enriched environment intervention group was significantly lower than in preweaning exposure group(P<0.01),and there was no statistically significant difference compared with the control group(P>0.05).3.Effects of enriched environment on morphological of dendritic spines in rats co-exposed to Pb,Cd and Hg.Golgi-Cox staining revealed that enriched environment increased the dendritic spine density in rats reduced by co-exposure to Pb,Cd,and Hg.In continuous exposure group and preweaning exposure group,the density of dendritic spines of hippocampus CA1,CA3,DG areas and prefrontal cortex were lower than in control group(P<0.01),and the density of dendritic spines in CA1,DG,and PFC areas in continuous exposure group was lower than in preweaning exposure group(P < 0.01).While the density of dendritic spines in the hippocampal CA1,CA3,DG,and cortical PFC areas of enriched environmental intervention group and preweaning exposure group were increased compared with the continuous exposure group,but it did not increase to the level of control group,preweaning exposure group increased to 91.38%,85.33%,84.46%,and 81.31% compared to control group,respectively,and enriched environmental intervention group increased to 93.85%,89.16%,81.72%,and 94.32% compared to control group,respectively.Moreover,the density of dendritic spines in the CA1,CA3,and PFC areas of enriched environmental intervention group was increased by approximately 2.71%,4.49%,and 16.01% compared with the unintervented preweaning exposure group,respectively.In addition,the enriched environment improved the dendritic spine morphology that was affected by co-exposure of Pb,Cd,and Hg,but did not return to the level of control group.The proportion of mushroom dendritic spines in hippocampal CA1 and CA3 and cortical PFC areas of continuous exposure group and preweaning exposure group was lower than in control group(P<0.05);in enriched environmental intervention group and preweaning exposure group,the proportion of the mushroom dendritic spines in the hippocampus and the PFC area were increased compared with the continuous exposure group,and the proportion of mushroom dendritic spines in hippocampal CA1 area of enriched environment intervention group was higher than in preweaning exposure group(P<0.05),and and the proportion of mushroom dendritic spines in PFC area of enriched environment intervention group was higher than in continuous exposure group(P<0.01).4.Effects of enriched environment on synaptic ultrastructure in hippocampal CA1 area.Transmission electron microscope showed that enriched environment improved the synaptic morphology and functional deficits caused by co-exposure of Pb,Cd,and Hg.In continuous exposure group and preweaning exposure group,the synaptic density was decreased,the thickness of PSD was thinner,the width of the synaptic cleft was widened and the length of synaptic active zone was shorter compared with the control group(P<0.01).Enriched environmental interventions increased the synaptic density and PSD thickness in hippocampal CA1 area compared with the preweaning exposure group(P<0.05),and preweaning exposure group and enriched environment intervention group had narrower synaptic cleft compared to the continuous exposure group(P<0.01),enriched environment intervention could restore the length of the synaptic activation zone but not restore to the level of control group,which was still lower than the control group(P < 0.05).5.Effects of enriched environment on Snk,SPAR mRNA and protein expression in hippocampus of rats co-exposed to Pb,Cd and Hg.RT-PCR and Western Blot revealed that enriched environment reduced the increase of mRNA and protein expression of Snk induced by co-exposure of Pb,Cd,and Hg.The expression of Snk mRNA and protein in the hippocampus of rats in continuous exposure group was higher than in control group(P<0.05),while the expression of Snk mRNA and protein expression in the hippocampus of preweaned exposure group and enriched environment intervention group was lower than in continuous exposure group(P<0.05).The mRNA and protein expression of Snk in the hippocampus in enriched environment intervention group had no significant difference compared with the control group(P>0.05).However,there was no significant difference in mRNA expression of SPAR in hippocampus of each treatment groups(P>0.05).On the other hand,immunofluorescence found that enriched environment upregulated the decreased expression of SPAR in rat hippocampus induced by combined exposure to Pb,Cd,and Hg.Compared with the control group,the expression of SPAR in the hippocampus of continuous exposure group and preweaning exposure group was decreased(P<0.01),the intervention of enriched environment increased the expression of SPAR in the CA1 region of the hippocampus compared with the natural recovery group(preweaning exposure group)(P < 0.05),but it did not increase to the level of the control group.6.Effects of enriched environment on the damage of hippocampal neurons in rats co-exposed to Pb,Cd and Hg.Enriched environmental intervention can improve hippocampal neurons injury induced by co-exposure of Pb,Cd and Hg.Transmission electron microscopy revealed that the chromatin in hippocampal CA1 area of the rats exposed to continuous exposure and preweaning exposure was aggregated into clusters,and the nuclear envelope remained intact,and the number of organelles also decreased,the mitochondria had severe swelling and the crista was broken and blurred.While the enriched environment intervention group was similar to the control group,which hippocampal neurons had smooth nuclear membranes,uniform chromatin distribution,and the number of organelles was increased,the mitochondria and the ridge morphology were more complete and clear.Flow cytometry revealed that the level of reactive oxygen species and apoptosis in the hippocampal neurons of continuous exposure group and preweaning exposure group were significantly higher than in control group(P<0.01).Compared with the naturally-recovered rats in the preweaning exposed group,the level of reactive oxygen species in the hippocampal neurons of enriched environment intervention group was reduced by 16.75% and the apoptosis level was decreased by 32.28%(P<0.01).Conclusion:1.The co-exposure of low concentrations of Pb,Cd and Hg can cause cognitive deficits in rats,especially the cognitive deficits induced by co-exposure of the three heavy metals is hard to be repaired naturally after weaning to adulthood.2.Enriched environment can improve the cognitive deficits in rats co-exposed to low concentrations of Pb,Cd,and Hg,which may be related to the improvement of synaptic plasticity and spine scaffold protein SPAR expression of rat,and which may be related to the decrease of oxidative stress in the hippocampal neurons.3.Enriched environment can improve the hippocampal neuronal injury in rats co-exposed to low concentrations of Pb,Cd,and Hg,which provide a scientific basis for further study of the repair of neurotoxicity damage induced by heavy metal pollutants. |
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