Effect And Mechanism Of Brain Damage Induced By Chlorpyrifos Inhalation Exposure

Objective:To establish a cognitive impairment model for CPF whole-body dynamic inhalation exposure on rat, standardize the particle size and concentration of CPF aerosol, simulating real dynamic acute inhalation exposure of CPF, and determine the median lethal time-integrated concentration(LCt50);To investigate changes of the performance of pathology and behavior,the expression of AChE, ACh, amino acid neurotransmitters and inflammatory response in different times and different brain regions after CPF inhalation exposure; To evaluate preventive effects on different types of anti-inflammatory drugs against CPF poisoning caused brain damage and cognitive impairment,and to discuss the relativities among the changes of neurotransmitters and inflammatory cytokines,and the brain damage after CPF poisoning. Methods:1. Establish a model for CPF inhalation esposure and determine the value of LCt50.By optimizing the aerosol parameters, the animal acute dynamic inhalation exposure environment of CPF was established. Absorption sampling-gas phase detecting technology was used to monitor the concentration of CPF in the whole-body dynamic-inhalation exposure cabin by exploring the relationship between the concentration, particle size of CPF aerosol and the CPF inhalation time in the exposure cabin by particle size detector. Using Bliss method, 50 specific SPF SD male rats were distributed into 5 groups randomly, with 10 per group. All the groups were allocated to the environment of CPF exposure, at different times of 5, 7, 10, 14, 20 hours. The symptoms and deaths of these SD male rats in different groups were recorded within the following 10 days;2. Changes in behavior and pathology in different brain regions after CPF exposure.Took 80 rats and used Morris water maze to perform training trial in order to detect spatial learning and memory ability.After training, taking 40 rats as the experimental group, SD male rats were distributed into 4 groups randomly, with 10 per group. All the groups were allocated to the environment of 0.4LCt50 CPF exposure;taking the remaining 40 rats as normal group, exposured to normal air under the same conditions. Took Morris water maze test and observed pathological changes respectively at 1d, 2d, 3d and 7d from each group after CPF exposure.3. Changes of AChE, ACh and amino acid neurotransmitters in different time after CPF exposure.280 SD male rats were distributed into 4 groups randomly, with 70 per group. Every groups were allocated to the environment of 0、0.2 LCt50、0.4LCt50、0.8LCt50 CPF exposure respectively. Got blood from caudal vein of 5 rats for each group.By using enzyme-linked immunoassay, hippocampus, striatum and frontal cortex were analyzed to determine if the levels of AChE had changed in 10 min, 3h, 8h, 1d and 2d, 3d and 7d after CPF exposure; in the meantime,by using high performance liquid chromatography(HPLC), three brain regions above were analyzed to determine if the levels of ACh, Glu,Asp,Gly and GABA had changed.4. Changes of inflammatory factorsvin different time after CPF exposure.30 SD male rats were distributed into 6 groups randomly, with 5per group. All the groups were allocated to the environment of 0.4LCt50 CPF exposure. By using real-time fluorescent quantitative PCR method, hippocampus and frontal cortex were analyzed to determine if the expression of IL-1β、TNF-α and NF-κB had changed respectively at 30 min, 8h, 24 h, 3d and 7d after CPF exposure;5. The protective effects of different types of anti-inflammatory drugs on cognitive impairment and brain damage caused by CPF exposure. 25 SD male rats were distributed into 5 groups randomly, with 5 per group, injecting tail vein by TNF-α specific antagonist(rhTNFR: Fc,3 mg/kg), the NF-κB specific antagonist(PDTC, 2 mg/kg), broad-spectrum anti-inflammatory drugs(ibuprofen, 10mg/kg) and normal saline respectively 30 min before CPF exposure. Taking out the whole right brain for HE staining,frontal cortex and hippocampus of the remaining left brain were analyzed to determine if the expression of IL-1β、TNF-α and NF-κB had changed in 3d after CPF exposure, in order to evaluate the preventive effects on different types of anti-inflammatory drugs against brain damage and cognitive impairment caused by CPF exposure. Results:1. In this experiment, aerosol particles(1.3μm) got close to numerical average(1.3μm) and geometric average(1.1μm), and conformed to the law of normal distribution according with the requires of OECD 403/433 acute inhalation toxicity experiment guidelines.The concentration of aerosols remained stable over time(160.6 ± 11.1mg/m3), coefficient of variation(RSD) within 7%, which illustrated that the concentration of aerosols were stable.Further we determined LCt50 of CPF is about 1654.3 mg/m3·h,and LCt95 is about 3015.6 mg/m3· h;2. Rats developed short-term spacial memory disorders within 1w after CPF inhalation exposure.Neurons in hippocampus and frontal cortex showed nucleus pycnosis, forming nodules or necrosis,while there was no obvious pathological damage in striatum area;3. In hippocampus and frontal cortex,the activity of AChE is significant declined in 30 min after CPF exposure compared with the normal(P < 0.05), then reaches the lowest point in 24 h,whereafter shows slow-rising.There are still significant differences in 3d(P<0.05).The activity of AChE does not get back to normal until 7d.While the activity of AChE in striatum varies slightly.It shows a significant decline in 24h(P<0.05),and gets back to normal in 3d after CPF exposure;4.The levels of ACh in different brain regions are related to the concentration of CPF.The levels of ACh reached a peak in 24 h, starting on a declining trend afterwards. There are still significant differences in frontal cortex and hippocampus in the 3rd day after CPF exposure(P<0.05), getting back to normal until the 7th day; However, the levels of ACh in striatum region of low dose group got back to normal in 2nd day after CPF exposure,while that of medium and high dose group got back to normal in 3d;5. The levels of amino acids neurotransmitters in different brain regions are related to the concentration of CPF. In high concentration group, EAAs in hippocampus appeared significantly increased(P<0.05) in 30 min after CPF exposure; In medium concentration group, Glu firstly appeared significantly increased(P<0.05) in 30 min, and reached its highest in 48h(P<0.01). While the Asp appeared a obvious increase(P<0.05) in 3h, reached its highest in 24 h; In low concentration group,the levels of Asp reached its highest(P<0.05) in 8h,while the levels Glu continously rised to its highest in 24h(P<0.05). In high and medium concentration group, the levels of EAAs in hippocampus still had a significant difference compared to normal(P<0.05) in 7d. While EAAs returned to normal levels in 1-2d in low concentration group. EAAs in frontal cortex had a similar variation, but got a half higher of those in hippocampus.The high levels of EAAs in frontal cortex last longer in low concentration group,returning to normal levels till 3d. The levels of EAAs in striatum area had a similar variation but changed little. The variation of IAAs was similar with that of EAAs.The only difference was that the levels of IAAs returned to normal in 7d in high and medium concentration group;6. The expression of IL-1β mRNA increased significantly in 30 min after CPF exposure(P<0.05) in hippocampus of rats, and the expression of TNF-α did the same in3h(P<0.05). While the expression of NF-κB mRNA changes slowly, increasing obviously until 8h after CPF exposure in hippocampus(P<0.05).These three inflammatory factors reached their highest peak in 48 h after CPF exposure, the expression of them in hippocampus still had a significant difference compared to normal(P<0.05) in 7d. The variation of these three inflammatory factors in frontal cortex was similar with that in hippocampus. The only difference was tha the expression of the three all increased significantly in 30 min after CPF exposure(P<0.05);7. PDTC improved cell damage in hippocampal CA1 area, as part of the neurons in the nucleus swelling hyperchromatic, individual associated with fiber entanglement, with a small amount of microglia infiltration; Damage is mitigated little in Ibuprofen group, characterized by a small neurons in the nucleus pycnosis hyperchromatic, accompanied by a small amount of fiber entanglement; Compared to PDTC and Ibuprofen group,RhTNFR:Fc can significantly improve cell damage in hippocampal CA1 area.The nucleus quality is clear, and only individual neurons in the nuclei were hyperchromatic,with basic normal cellular structure; PDTC and Ibuprofen reduced damage degree in frontal cortex area,with part of the tangles, hyperchromatic cells;Ibuprofen could reduced damage degree somehow,but there were still focal pyknotic hyperchromatic cells,with disorder arranged, forming the trend of the formation of nodules. Conclusion:The animal acute dynamic inhalation exposure of CPF was established in this experiment. Rats appeared short-term spacial memory disorders within 1w after CPF exposre.In the first few hours, the rapid accumulation of ACh in the affected brain regions such as the hippocampus and frontal cortex would cause serious toxicity of excitatory and the dysfunction of cholinergic neurons, damaging the integrity of the cholinergic pathway in cells. The toxicity of excitatory also can break the balance between th activity of the Glu and GABA, causing neuronal excitatory toxic injury and inflammation. The cholinergic neuronal excitability toxicity would lead a wide range of intracellular edema, nucleus pycnosis, excitability toxicity caused by EAAs increasing, nerve inflammation and stress reaction caused by up-regulated expression of the inflammatory factors. All the facters above can bring about various negative effects, inducing the loss and death of brain neurons. Interventions based on the inflammatory reaction may be new ways of prevention to brain damage caused by CPF.