The Preliminary Study Of Urban Road Sur -Rounding Atmospheric Fine Particles On The Neurotoxicity In Rats

ObjectivePMi (aerodynamic equivalent diameter< 1 microns, PM1) accounts for most of the quantity of PM2.5 particles, is the important element of the ash haze weather.PMi consists of toxic substances such as polycyclic aromatic hydrocarbons、heavy metals and so on is a mixture,characterized by small size、light quality、quantity et al, easily absorbed into the blood stream, and closely related to people health. This study infected rats with collected PM1, observed the toxicity of PMi to central nervous system, and preliminary discussed PM1 s toxic mechanism on CNS, aiming to provide the experimental basis and theoretical reference for urban air pollution effects on the CNS related exposure population research.Method1. PMi samples collectedThis study collected atmospheric fine particles with the Finnish Dekati DLPI-impact type multistage particles sampler at the city’s main traffic road distanced about 1 meter (sampling mouth distance the ground is 1.5 m) from November 16,2013 to May 12,2014. Polycarbonate membrane filter producted by Whatman company was selected as sampling filter membrane, setted sampling flow to 301/min.Replacement of a membrane filter every 24 h, rain and snow day stop sampling.2. PM, infected50 SPF male SD rats were randomly divided into 5 groups:blank control group, saline group(equal volume of distilled water), three PM1 exposure groups(low dose group (1.5 mg/kg), middle-dose group (7.5 mg/kg), high dose group (37.5 mg/kg)),10 rats in each group. After ether anesthesia, PMi was given to rats by tracheal drip in the volume of 2.5ml/kg.bw for 3 times,1 time two day.3. Observation of the general stateEach group general condition of rats was observed everyday, recorded weight on a regular basis, and detected the viscera coefficient of whole brain, the cortex and hippocampus.4. Observation of central neurotoxicity effects4.1 Morris water maze was used to determine the learn and memory ability of rat. In the Morris water maze experiment, the escape latency, the total distance and the average crossing number were recorded.4.2 Two rats were randomly selected from each group at the end of the Morris water maze experiment. Then HE staining slices were prepared to observe the changes in cerebral cortex and hippocampus of rats in all groups. c.Enzyme-linked immunosorbent assay (ELISA) method was used to detect single amine neurotransmitter norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in hippocampus.5. Investigation of the central neurotoxic mechanism of PMi5.1 At the end of the experiment, blood samples and cerebral cortex samples were get after the rats were killed to prepare the serum samples and cerebral cortex homogenates. Oxidative stress Kit was used to determine the T-SOD activity, GSH-Px activity, total antioxidant capacity (T-AOC),GSH and MDA.5.2 Part of the cerebral cortex of rat was taken to prepare cell suspension. ELISA method was used to determine the concentration of IL-1β、IL-6、TNF-α in cerebral cortex. The expression of nuclear factor kappa B in cerebral cortex and hippocampus was determined by immunohistochemical staining method.5.3 TUNEL method was used to observe the neuronal apoptosis of cerebral cortex and hippocampus in all groups.Result1. The effects of PMi infected on the rats weight and viscera coefficient of whole brain, the hippocampus, cortex1.1 The general condition of rats in all groups was well. Sustained growth in the control group weight during test, each weight fluctuate infected group, saline group and the low, middle dose group infected 5 or 7 days after infected body weight was significantly lower than the control group (P<0.05), other groups of animal body weight showed no significant changes when compared with the control group and the saline group (P>0.05).1.2 The brain organ coefficient of high-dose group was decreased significantly when compared with the control group and the saline group (P<0.05), the brain organ coefficient of medium group was decreased significantly when compared with the control group (P<0.05).Other indexes of all groups showed no significant changes when compared with the control group and the saline group (P>0.05).2. Central neurotoxicity of PMi2.1 In the place navigation test, compared with the control group and the saline group, the escape latency of medium and high-dose group was higher in the third and fourth day (P<0.05); the total distance of middle-dose and high-dose group were higher than the control group and the saline group in the third and fourth day (P<0.05); the other groups’ total distance and escape latency showed no significant changes in all four days. In the spatial probe test, the average crossing number each infected group was lower than the control group (P<0.05),the average crossing number high-dose group was lower than the saline group (P<0.05), the surrounding distance medium and high-dose group were lower than the control group and the saline group(P<0.05); but there were no significant changes in other groups with other indexes.2.2 From the pathological slices we can not see obvious abnormity in the control group and the saline group, characterized by different brain regions uniform color,structure dense and clear,most of nerve cells present as oval, cone and irregular shape, the nucleus blue dye, outline clear.Each infected group rats cortex and hippocampus neurons appear different degree of pathological changes, visible cells arranged disorder, the decrease in the number of neurons, nucleus pycnosis, chromatin dissolved and so on.As the canister to higher doses, more abnormal tissue can be seen,and pathological changes more serious.Nerve cells’s edema and vacuolization can be seen in high dose group,liquefaction necrosis in the gate region also can be seen in hippocampus.2.3 According to the determination of rat hippocampal single amine neurotransmitter, 5-HT and DA content in high dose group were significantly lower than control and the saline group (P<0.05);Content of NE in each infected group were significantly higher than that of control group (P<0.05), the content of NE in the medium and high dose group was also significantly increased compared with saline group (P<0.05), the rest of the indicators in each group compared with control group and saline group had no significant change (P>0.05).3. Central neurotoxic mechanism of PM13.1 We determined the indexes of oxidative stress in cerebral cortex. Compared with the control group, the activity of T-SOD was decreased in high-dose group (P<0.05); the content of MDA was increased in each group (P<0.05); the content of T-AOC was decreased in middle-dose and high-dose groups (P<0.05); other indexes in the rest groups showed no significant changes (P>0.05). Compared with the saline group, the activity of T-SOD was decreased in medium and high-dose group (P<0.05); the content of MDA was increased in medium and high-dose group (P<0.05);the content of T-AOC was decreased in middle-dose and high-dose groups (P<0.05); but there were no significant changes in other indexes (P>0.05). The indexes of oxidative stress in serum showed that the activity of T-AOC was decreased in each group when compared with the control group and the saline group(P<0.05); the content of MDA was increased in each group compared with the control group (P<0.05), the content of MDA was also increased in high-dose group compared with the saline group (P<0.05);the content of GSH was increased in medium group compared with the saline group (P<0.05),the content of GSH was increased in high-dose group compared with the control and saline groups (P<0.05);the content of GSH-Px was increased in high-dose group compared with the control and saline groups (P<0.05);but the other indexes in the rest groups showed no significant changes (P>0.05).3.2 According to the results of determination of inflammatory cytokines in the rats cortex, the content of TNF-a were significantly increased in medium and high dose groups compared with control group and saline group (P<0.05), the content of TNF-a in low dose group was also significantly increased compared with the control group (P<0.05); The content of IL-1β in high-dose group was also significantly increased compared with the saline group (P<0.05);The rest of the group the indexes had no significant change (P>0.05).Immunohistochemical results showed that the expression of NF-kappa B were significantly increased in cerebral cortex and hippocampus in medium and high dose groups compared with control group and saline group (P<0.05).3.3 TUNEL results showed that TUNEL-positive cells were significantly increased in the cerebral cortex and hippocampus of medium and high dose groups when compared with the control group and the saline group (P<0.05).Conclusions1. Under the condition of this study,PM1 can make the spatial learning and memory abilities of rats decreased, and then affects the central nervous system function by tracheal drip infected.2. Under this test dose,PM1 exposure can cause rat cerebral cortex and hippocampus tissue pathological damage.3. PM1 exposure can cause hippocampal tissues single amine neurotransmitter NE, DA and 5-HT content change.4. PM1 exposure can enhance oxidative stress in the serum and cerebral tissues of rats.5. PM1 can cause rat cortex cell inflammatory cytokine TNF-a, IL-1 beta content changes,also can cause the NF-kappa B expression change.6. PM1 exposure can induced neuronal apoptosis in the cerebral cortex and hippocampus tissue of rats.To sum up,PM1 infected can cause central nervous system of rats functional and pathological change, single amine neurotransmitter in the brain in a significant change in this study, lead to the traumatic change mechanism may be associated with brain peroxidation enhanced, inflammatory factors mediating and release, and neuronal apoptosis increased.