Effects Of Chlorpyrifos On The Related Brain Area Of Dopaminergic Neurons During Embryonic Development

Chlorpyrifos(Chlorpyrifos,CPF),a widely used organophosphorus pesticide in the environment,has a certain effect on embryonic neural development toxicity.The dopaminergic system of the brain is sensitive to organophosphorus pesticide exposure and associated with a variety of cognitive and emotional disorders,at present,there are few reports about the study of embryonic brain tissue cells.The subject was explored the effect of chlorpyrifos to nerve cells on the related brain area of dopaminergic neurons,furtherly,the influence of regional morphology by three-dimensional structure,to observe the toxicological effects of chlorpyrifos on macro and micro levels.Firstly,the effects of exposure to chlorpyrifos of the nerve cells in striatum,ventral tegmental area and substantia nigra:ICR mice were exposed to 0,5mg/kg CPF during embryo day 7.5-11.5,to obtain the timing embryos of E14,E16,E18.Using the Olympus IX71 Microscopy to count the number of the nerve cells of striatum,ventral tegmental area and substantia nigra after locating brain tissue sections and Nissl staining for statistical analysis,and the results discover that:(1)The striatum region:The neurons in the striatum area were showed round,oval,and had cell bodies,which had large round nuclei in the central,glial cells were showed granular form with the shape of flat,triangle,star;under the treatment of chlorpyrifos,there was no significant change on cellular morphology,but the cell density compared with the normal group,slightly loose.After the treatment of chlorpyrifos,the neurons of E14 were decreased by 12.78±0.5%,the glial cells were reduced by 31.93±3.0%;the neurons of E16 were decreased by 11.09±1.6%,the glial cells were reduced by 26.98±1.2%;the neurons of E18 were decreased by 11.03±0.5%,the glial cells were decreased by 22.02±1.7%.(2)The ventral tegmental area:In the ventral tegmental area,the cell bodies and projections of neurons could be observed,even the dendrites;glial cells showed the round and shuttle shape.Compared with the normal group,the cells did not change significantly as a whole after chlorpyrifos treatment.After chlorpyrifos treatment,the glial cells of E14 were reduced by 34.25±2.9%;the glial cells of E16 were reduced by 27.92±4.0%;the glial cells of E18 were reduced by 12.0±1.1%,but the neurons were not statistical difference.(3)The substantia nigra:Neurons in the substantia nigra are oval or conical shape,neurites is obvious,nuclear staining is weak;the glial cells have neurites,and the morphology is spindle shaped and round,but do not have dendrites and axons.There was no significant difference in cell morphology after chlorpyrifos treatment,but the cells density was slightly looser compared with the normal group.After chlorpyrifos treatment,the neurons of E14 were decreased by 9±1.40%,the glial cells were reduced by 36.47±4.5%;the neurons of E16 were decreased by 10.09±1.7%,the glial cells were reduced by 32.79±3.0%;the neurons of E18 were decreased by 7.29±0.6%,the glial cells were decreased by 23.52±3.4%.Overall,embryonic period exposures to chlorpyrifos had no obvious effect on cell morphology,but the number of glial cells decreased obviously in three regions of striatum,ventral tegmental area,substantia nigra area,and the number of neurons reduced in the substantia nigra and striatum,while the number of neurons in the ventral tegmental area had no obvious effect,These two kinds of cells,the effects of chlorpyrifos on glial cells was stronger than that of neurons.Secondly,the effects of chlorpyrifos on the dopaminergic neurons in embryonic brain:The effects of exposure to chlorpyrifos on the dopaminergic neurons in the brain regions were studied by TH immunofluorescence staining,the results found that:1)The ventral tegmental area: There are a large number of dopaminergic neurons in the ventral tegmental area,which are arranged in an irregular distribution.The dopaminergic neurons present an oval shape,and the size of the cell body shows a moderate size.There was no significant change in the morphology of dopaminergic neurons after chlorpyrifos treatment.The cells labled with DAPI decreased by 9.50 ± 0.8% after chlorpyrifos treatment,but there was no significant difference in the number of dopaminergic neurons and TH/DAPI.2)The substantia nigra: The substantia nigra dopaminergic neurons density concentrated,The arrangement is scattered and the neurons are pyramidal.There was no significant change in the overall morphology of the cells treated with chlorpyrifos,but the arrangement was slightly scattered compared with the normal group.The cells labled with DAPI decreased by 14.24 ± 1.5% after chlorpyrifos treatment,the dopaminergic neurons were reduced by 11.51±0.5%,but there was no significant difference in the ratio of TH/DAPI.On the whole,embryonic period exposures to chlorpyrifos had significant inhibitory effects on the cells labled with DAPI in the ventral tegmental area and substantia nigra,and the dopaminergic neurons were also inhibited to a certain extent.But the ratio of TH/DAPI didn’t change,indicating that the inhibitory effect of chlorpyrifos on dopaminergic neurons was nonselective.Thirdly,the effects of chlorpyrifos on the three-dimensional structure of embryonic striatum:The serial sections of the striatum of E16 were photographed by microscopy.Used Photoshop CC,Image Pro Plus,and 3D Doctor to reconstruct the three-dimensional structure of the striatum,and analysis correlation parameter.The results discovering,the volume of E16 striatum was 0.43 mm3 in Control group,and 0.42 mm3 in CPF group;the surface area of Control group was 13.43 mm~2,and 9.40 mm~2 in CPF group.In summary,we found that embryonic period exposures to chlorpyrifos,which had a significant influence on nerve cell micro level.But in the macro morphology,the effects of chlorpyrifos was tiny on the overall regional structure.