Effects And Mechanism Of Excessive Lead Exposure During Perinatal Period On Sleep/Wakefulness In Adult Mice

Part One Effects of ealy life lead exposure on sleep architectureStudy Objectives: An animal model of lead exposure mouse was established and testified.The effects of exposure to different concentrations of lead during the period from 2 weeks before embryonic formation to 1 month after delivery were observed for the influence of long-term sleep disorder in mice.Total sleep time,sleep latency,daytime awakening,and sleep switching ability of the mice were used to determine whether lead exposure during peri-embryonic period in mice caused sleep disturbances in adult mice and was analyzed by sleep electroencephalogram.Design: 1.Six adult female J:DO mice were randomized into three groups using the random number table method.They were 0 ppm group,30 ppm group,and 300 ppm group.Female mice were started to administer each group of lead-containing solutions at a corresponding concentration two weeks before mating,and then one adult male J:DO mouse,normal drinking water and fed male mice,was mated separately in their cages until the female mice became pregnant.Male mice were divided into cages.The female mice were continuously given a corresponding concentration of lead in each group during pregnancy and continued until the females had delivery.After that,the females continued to drink the lead-containing solution and then breast-feeded the young female until 4 weeks later when the mice could eat independently.Mice and mother rats were housed in separate cages.Thereafter,the young mice were given regular drinking water and food.2.Blood samples were taken at random from 0,30,and 300 ppm three groups of fetal leadexposed young mice at different concentrations to detect blood lead levels during infancy.3.When the mice entered adulthood(6 months of age),the head and neck muscles of 3 groups of mice were implanted into 24 hours of continuous sleep EEG and EMG signals were collected after debugging the sleep recording device.4.The results of the sleep recording were automatically analyzed using the algorithm of version 3.0 of the Windows version of Sleep Sign for Animal software,and were manually corrected frame-by-frame.After the calculated results were summarized,the subsequent statistical analysis was performed.Results: 1.Residues of lead in mice:(1)Blood lead concentrations at 1 month of perinatal exposure to lead in mice.Group comparison: Compared to the 0 ppm control group(6.0 ± 7.6 ppm),blood lead concentrations in the 30 ppm group increased significantly(125.8±13.9ppm,p<0.0001),300 ppm blood lead concentration was significantly increased(418.9±35.2ppm,p<0.0001);(2)lead exposure in adult rats(6 months of age)in vivo blood system lead remnants and childhood comparison: There was no significant change in lead concentration in adult 0 ppm group(<0.01 ppm,p = 0.9887)compared with the monthly 0 ppm group mice(6.0 ± 7.6 ppm);compared to the 1 month old 30 ppm group(125.8 ± 13.9 ppm),The blood lead concentration in the adult 30 ppm group was significantly lower(<0.01 ppm,p<0.0001);compared with the one-month 300 ppm group(418.9±35.2ppm),the blood lead concentration in the adult 300 ppm group was significantly reduced(0.0376±0.01 ppm,p<0.0001).(3)Comparison of blood lead residual concentrations in adult mice after maturation(6 months of age): Compared with the 0 ppm group(<0.01 ppm),the lead concentration in the 300 ppm group was significantly higher(0.0376±0.01 ppm,p<0.001).;(4)Liver lead residue in mice after maturation(6 months old)comparison between groups: Compared to 0 ppm group(0.1 ppm),lead concentration in liver of 300 ppm group(0.8446±0.48 ppm,p<0.0001)significantly increase;(5)Adult mouse(6 months old)long bone lead concentration between groups: Compared with 0 ppm group(0.1 ppm)(0.1 ppm)),lead concentration in the 300 ppm group of bones was significantly higher(57.38±25.43 ppm,p<0.001);2.Changes in sleep structure after lead exposure in mice:(1)Changes in wakefulness: Compared with 0ppm in the control group(44.5±13.7 min),the proportion of awakening of ZT2-4 in the 30 ppm group was lower after lights-on(65.2±13.5min,p<0.05).Compared with the control group(53.7±17.3 min),the proportion of awakening in the ZT8-10 decreased in the 300 ppm group(75.0±16.3min,p<0.05);Compared with the control group(79.0±12.4 min),300 ppm group awakening proportion decreased in ZT12-14(101.0±10.5 min,p<0.05);2NREM sleep maintenance ability changes: compared with the control group Compared with ZT8-10(58.7±13.1 min)and ZT12-14(38.1±11.5 min),the NREM sleep volume decreased by ZT8-10(39.8±15.7 min,p<0.05)after light turn on in the 300 ppm group,ZT12-14(16.6±8.9 min,P<0.05).3 Changes in REM sleep volume after light off: Compared with the control group(25.9 ± 8.7 min),REM sleep increased significantly in the 300 ppm group(37.4 ± 6.9 min,P < 0.05).4 Changes in sleep state transition ability: Compared with the control group(273.8±44.8 times),the number of conversions from NREM sleep to wakefulness in the 300 ppm group was significantly reduced(204.3±70.2,P<0.01);compared with the control group(290.5±)42.7 times),the number of conversion from awakening to NREM sleep in the 300 ppm group also decreased significantly(239.6±73.9,P<0.05).5 Short-term awakening(<16s)The total length of time after lighton and turn-off time changes: Compared with the control group after turning on the lights(210.7±66.8 times),awakening of 300 ppm at <16s was significantly reduced(103.1±56)(P<0.0001);After turning off the lights,compared with the control group(149±21.3),the 300 ppm awakening segment at <16s decreased(103.1±56 times,P<0.0001).Conclusions: 1.Lead exposure in Perinatal Period exposes the mouse's blood lead levels much higher than in adult rats;compared with the control group,mice exposed to 300 ppm lead during the Perinatal Period still reserved more in the blood,liver and bone after maturation.The amount of lead accumulated in the bone is significantly higher than that in blood and liver.2.Compared with the control group,mice exposed to lead significantly increased their awaken time after light-on,and decreased REM sleep after light-off.3.There was no significant difference in the total amount of sleep during lighton period,but there was a disorder in the ability to switch sleep state(between NREM and Wake).4.Compared with the control group,the total number of short-term arousal segments(<16s)decreased during the whole day(including the light-on and light-off periods)in lead-exposed mice,indicating that lead-exposed mice have reduced alertness during both sleep and wakefulness,and have reduced responsiveness to external stimuli.Part Two Effects of early life lead exposure on sleep/wake neuronStudy Objectives: To determine whether lead concentrations in the brains,mice are present at the age of 6 months and to measure their relative concentrations,and to combine the function of the corresponding brain regions and their association with normal sleep and wakefulness.To determine the objective of this experiment and study the brain regions,three-dimensional space counting,morphological observation and quantitative detection of the sleep/wakeful neurons in their corresponding nuclei were used to determine whether injury and apoptosis in sleep/wake neurons happened or not.Design: 1.Continue with the first part,young rats were given normal doses.Drinking water,feeding and raising until 6 months of age.One mouse was randomly selected from three groups.The lead brain deposition level in brain tissue was detected by laser ablation spectroscopy in the main brain slices to determine the main enrichment area of lead.And the relative concentration,and compare the concentration differences between the three groups.2.According to the association of lead-rich regions with known sleep-regulating regions,the relevant brain regions would be targeted and be further studied.3.Five mice were randomly selected from three groups of mice.The total number of Orinxin neurons in the lateral hypothalamus of each group of lead poisoning mice was evaluated using Stereo Investigator software under light microscope.Five mices were randomly selected and RTPCR was used to detect the expression level of Orexin in the LH region.The expression of Orexin was further verified at the RNA level.Results: 1.Compared with the control group(3944.8±249.4),the number of orthin neurons in the hypothalamic lateral area of peri-implantation lead-exposed mice was significantly decreased in the 30 ppm group(3515.4±134.4,P<0.05).300 The group of ppm decreased significantly(2735.0±153.9,P<0.01).2.The density of Orexin neurons in the LH region was significantly decreased in the lead exposed group under the light microscope compared with the control group.RT-PCR showed that in the lateral hypothalamus region,compared with the control group(2.3±1.0),the 30 ppm group(0.5±0.3,P<0.001)Orexin m RNA expression level showed a significant decrease,300 ppm group(0.5± 0.4,P<0.001)decreased significantly.Conclusions: 1.Excessive lead exposure during Perinatal Period can lead to excessive lead accumulation in the brain of adult mice(6 months old).Lead is mainly deposited in the mouse cortex,lateral hypothalamus,and hippocampus.2.Excessive lead exposure during embryonic period caused the number of Orexin-positive neurons in the LH region decline significantly after mice matured,and the density of Orexin neurons decreased,suggesting that lead exposure during Perinatal Period leads to long term sleep disordered.Part Three Exploring the mechanism of sleep/wake neuron damage due to lead exposureStudy Objectives: To detect content of Tau protein in the lateral hypothalamus and the p-tau protein content in the hypothalamus.To explore mechanism of lead exposure induced damage of sleep/wake neurons and its relationship with Tau.Design: 1.Periembryonic exposure to lead exposure mice were divided into 3 groups(0ppm,30 ppm,300ppm).Five mice were randomly selected from each group.After anesthesia was performed,the lateral hypothalamic area p-Tau(AT8)was observed by immunofluorescence.The expression of Orexin and the fluorescence intensity were analyzed using image j software to determine whether or not tau protein was deposited and its severity in orexin neurons.2.The co-expression of microglia(IBA-1)and Orexin was detected by immunofluorescence,and the number of microglial cells surrounding Orexin neurons in lead-exposed mice during embryonic period was determined.3.The expression of phosphorylated Tau protein(Ser-202)and microtubule-associated protein tau(MAPT)in the hypothalamus region was determined by Western-blot.Results: 1.Immunofluorescence semi-quantitative analysis results: Compared with the control group(0.07±0.02),p-Tau(AT8)expression in the orexin neurons in the lateral hypothalamus of 30 ppm lead exposure group was significantly higher(0.78±0.30,p<0.05),300 ppm The expression of p-Tau(AT8)was significantly increased in lead exposure group(2.23±0.54,p<0.0001).2.Compared with the control group(0.621±0.030/pixel),the number of microglial cells in the LH region of the 300 ppm lead exposure group was significantly reduced(0.203±0.016/pixel,p<0.001).3.WB results: Compared with the control group(7.21±1.03),there was no significant difference in the Tau(MAPT)protein in the lateral hypothalamus of the 300 ppm lead poisoning group(6.92±1.53,P>0.05);Compared with the control group(0.79±0.18),the amount of phosphorylated Tau(Ser-202)protein in the 300 ppm lead exposure group(1.70±0.43,p<0.01)was significantly higher in the group.Conclusions: Exposure to lead caused excessive Tau protein accumulation in Orexin neurons,accompanied by a decrease in the number of microglial cells in the LH region,suggesting that lead exposure during the embryonic period may lead to bioactive phosphorylated Tau in Orexin neurons in adult mice.Abnormal protein deposition,the ability of the brain to scavenge poisonous material weakened,which may explain Orexinergic neuron damage in lead explosed mice.