Effects Of Early Exposure To Chlorpyrifos On The Development Of Hippocampus In Mice

Animal embryos are susceptible to environmental toxicants,such as organophosphorus peptides.Hippocampus is a major component of the brains responsible for cognitive behavior.In this study,we investigated the effect of chlorpyrifos(CPF)exposure to the development of hippocampus.We injected CPF into mice embryos at E7.5-E11.5 at a dose of 1mg/kg/day or 5mg/kg/day and checked variation of several indicators.The activity of acetylcholinesterase(AchE)in the brain was measured both in male and female mice embryos.The number of neurons and glial cells were counted at E12,E15,and E18,respectively.Finally,we constructed the three-dimensional structure of the hippocampus region by continuous tissue slicing and 3D reconstruction software.Our results showed that the activity of AchE was affected by treatment of CPF during E7.5-E11.5 in the embryonic brain.Compared with control E12 samples,the AchE activity decreased to 77.8 ± 6.55% and 80.48±6.85% in female and male mice embryo,respectively,when treated with1mg/kg/day CPF.The values were decreased to 62.95±6.45% and 66.52±5.96% when treated with 5mg/kg/day CPF.To E15,compared with the control group,the AchE activity decreased to 87.10±8.70% in female and87.10±8.70% in male mice embryo when 1mg/kg/day CPF was injected to mother mice.The activity in female and male mice embryo treated with5mg/kg/day CPF were down to 78.00 ± 5.69% and 78.00 ± 5.69%,respectively.By E18,we found that the AchE activity in 1mg/kg/day CPFtreated female and male embryo were 99.79% ± 13.78% and 95.06 ±23.31% of control group.And in 5mg/kg/day CPF treated embryos,the values were 85.46 ± 9.03%(female)and 95.06 ± 23.31%(male),respectively.In all three groups,no significant difference in the activity of AchE was observed between female and male embryos(p>0.05 for each group).From these results,we got know that embryo exposure to CPF could inhibit the activity of AchE,and the influence could be restored over time.Furthermore,this kind of inhibitory effect has no gender selectivity.The effect of CPF exposure to the cell number of neurons and glial cells in embryonic hippocampus were as follows:(1)when embryos were exposed to 1mg/kg/day,significant reduction of both neurons(p<0.05)and glial cells(p<0.05)were observed in E12 samples.By E15,the number of both neurons and glial cells in CA1 region reduced significantly,while in CA3 region,only glial cells reduced significantly.To E18,neither glial cells nor neurons showed statistical difference between CPF-treated and non-treated samples,either CA1,CA3 or DG region.(2)Under the treatment of 5mg/kg CPF,the number of glial cells and neurons decreased significantly in hippocampus primordium at E12.By E15,the number of glial cells(p<0.01)and neurons(p<0.01)decreased significantly in the CA1 and CA3 region.By E18,the number of glial cells(p < 0.01)and neurons(p<0.01)still had decrease in the CA1,CA3 and DG region.(2)Under the treatment of 5mg/kg CPF,the number of glial cells and neurons decreased significantly in hippocampus primordium at E12.By E15,the number of glial cells(p< 0.01)and neurons(p< 0.01)had a significant decrease in the CA1 and CA3 region.By E18,the number of glial cells(p<0.01)and neurons(p<0.01)still had decrease in the CA1,CA3 and DG region.Finally,we prepared continuous slices of the E18 hippocampus regionand reconstructed the three-dimensional brain structure by using 3D-doctor software.Data analysis showed that the volume of hippocampus and coronal 7-19 in control group were 0.36 um~3 and 5.31 um~3,respectively,while they are 0.35 um~3 and 5.24 um~3 in CPF-treated group.Therefore,CPF had no significant effect on the three-dimensional morphology of the hippocampus.Taken together,these results indicate that,(1)The inhibition of Ach E activity caused by CPF can be restored at certain extent.(2)The effect of CPF toxicity on the embryo had no selectivity to gender.(3)CPF had an inhibitory effect on glial cells and neurons in the embryonic hippocampus,which can be sustained to E18 days or more.(4)CPF exposure had no significant effect on the macroscopic structure of hippocampus.