| With the rapid development of modern industrialization,the lead has become an important industrial poison and environmental pollutants,and it can cause injury to systems and organs in human body.In recent years,the effect of lead on children's nervous system,especially to children's developing nervous system,is very conspicuous.Thus,the hazard from lead has attracted wide attention from scholars all over the world,and become a research hotspot.Because of special developing and metabolic characteristics,lead neurotoxicity to children is very dangerous,causing the problems of mental,neurological behavior,memory and cognitive ability,and so on.The study showed that in the case of the long-term exposure to environmental of lead pollution,the lead was easyly accumulated in the brain and lead to chronic irreversible neurologic injury.It is confirmed that the amygdala is selective accumulation area of lead.The amygdala is one of the important structures in central nervous system.In recent years,many researches were performed on structure and function of amygdala,and it integrates a variety of sensory information,participating in emotional,cognitive and behavioral regulation,especially cognitive function and it becomes currently hot spots of research.The studies on amygdala from anatomical level in the past to the molecular level nowaday,mainly focused on mechanisms of receptors.It has been confirmed that estrogen receptors distribute in amygdala,playing a neuroprotective function after combining to its ligand estrogen and participating in the cognitive ability regulation.The effects of lead exposure on cognitive function have been intensively studied over the past decade,but less attention has focused on its impact on amygdala.This study aimed to investigate the effects of lead on the amygdala of developing rats,as well as the underlying molecular mechanisms responsible for these effects.Objectives1.To construct the model of lead exposed rats,and to observe the effects of lead exposure on blood lead and lead content in the amygdala of rats at different time,and to provide evidence for further study on the effect of lead on the amygdala.2.Electron microscope was used to observe the effect of lead exposure on the ultrastructure of amygdala in different developmental stages of rats,and to observe the effect of lead exposure on the cognitive ability of rats by Morris water maze test.3.Using real-time PCR and western blotting to test the expression level on mRNA and protein of ER ? and ER? in the amygdala through lead exposed groups in different exposing time(30 days,60 days and 90 days)in the developmental rats and non lead exposed group Eventually we provide theoretical basis for studying the effects of lead exposure bn the amygdala in developmental rats.Materials and Methods1.Experimental animals1.1 Subjects30 Healthy adult Wistar rats were selected(Provided by the animal experimental center of Zhengzhou University Medical College,animal Certificate No.Day05035),weight:240 ± 20 grams,including 10 males and 20 females rats.After 7 days adaptive feeding in a cage,in accordance with the female:male = 2:1,in the next morning to check the vagina contraceptive suppository,or vaginal secretion smear microscopy to determine the pregnancy station,20 females rats were conceived and fed in single cages.There were 120 3-week-old male Wistar rats were selected as the research objects,weighing about 60-80g.1.2 Establishment of animal model and grouping120 Male Wistar rats were divided randomly into 4 groups by sortition method,with 30 rats in each group,including the control group(DayO group)and different time lead exposure group(Day30 group,Day60 groupand Day90 group).strictly controlling the feeding environment,to free water given to the control group of rats without lead deionized water,three lead exposed rats divided into drinking 0.2%lead acetate solution 30 days,60 days and 90 days.The day 0 group and day 30 group in 30 days were conducted.According to experimental requirement,day60 and day90 group in 60 days and 90 days for experiment,respectively.1.3 Feeding conditionExperimental animals were at room temperature for 20 to 25 degrees,good ventilation,maintaining feeding circadian ratio of 1:1 clean environment,strict disinfection and lead pollution controlled.Conventional breed of female rats during pregnancy and lactation.The experimental subjects were divided into groups to feed according to the experimental requirements.The clean feed is provided by the experimental animal center,Medical College of Zhengzhou University.2.Methods2.1 Draw materialsTake the blood:20 rats of each group were randomly selected,respectively,the tail of the rats followed by soap water and deionized water purification,then dip in the warm water about 40 to 50? for about 5-10 minutes,in order to make the tail vein vascular filling,with the tail of the rats with alcohol cotton wipe,with 5ml of sterile injection extraction tail vein blood,let the blood dripping into and ethylene diamine tetraacetic acid disodium salt(containing tetracetic acid,EDTA anticoagulant tubes,and into the-80 degrees refrigerator preservation for blood lead level.Isolated amygdaloid tissue:after blood samples were collected immediately on rats,decapitated brain on ice quickly stripped of brain tissue,frozen on ice cold physiological saline,and then carefully isolated amygdaloid tissue.Each rat out side of the amygdala were fixed,rinsed,embedding,sectioned,and dyed.At last,observ the ultrastructure of amygdala through transmission electron microscope.On the other side of the amygdala are packed in clean vials,sealed into the liquid nitrogen,and then stored to-80 degrees refrigerator preservation reserve.For testing lead content in the amygdala and testiing of mRNA and protein expression of ER a and ER?.2.2 Content of lead in rat blood and amygdala was determined by graphite furnace atomic absorption spectrometry.2.3 The ultrastructure of amygdala in rats was observed under transmission electron.2.4 Morris water maze test was used to detect the spatial cognitive ability of rats in each group.2.5 Using Real-time PCR to detect the mRNA expression level of ER a and ?in the amygdala of the 0d,30d,60d and 90d.2.6 Western blotting method was used to detect the protein expression levels of ER a and ? in the amygdala of,Od,30d,60d and 90d.2.7 Statistical analysisUsing SPSS 21.0 statistical software package for statistical analysis.Morris water maze escape latency with repeated measures analysis of variance analysis,the rest of the variables with single factor variance analysis,when there are differences in groups of samples,LSD method was used to analyze the difference between the two groups.Spearman rank correlation was used for correlation analysis,the standard of inspection is a = 0.05.Results1.Determination of lead in blood and amygdalaCompared with the control group,the content of blood lead and amygdala lead levels in three lead exposure groups(Day30,Day60 and Day90)were significantly higher than those in the control group,were significantly different(P<0.05);and with lead exposure time prolonged,blood lead levels and the lead contents in amygdala of three groups presented increasing trend and between groups were compared with significant difference(P<0.05);Spearman rank correlation analysis is used to analyze the relation of the blood lead level and lead content of amygdaloid of Day30,Day60 and Day90 group after exposure to lead.Correlation coefficient r = 0.66,v=58,P<0.01,that is after lead exposure rats blood lead level and amygdaloid tissue lead content was significantly and positively correlated.2.The ultrastructure of amygdala was observed under electron microscope.It can be seen that lead exposure rats neurons of nuclear membrane depression,cytoplasmic loose change;amygdala presynaptic vesicles increased,intensive is deep dyed,increased neuronal activity;amygdala swelling of mitochondria,mitochondria cristae disappeared and mitochondrial disintegration is also visible,the amygdala in the sheath is like demyelination changes.3.Results of Morris water maze testCompared with the normal control,the escape latency of the rats in lead exposure group was longer than that in the control group during each training day,and was positively correlated with the lead exposure time.4.Effect of lead exposure on the expression level of mRNA and protein of ER a and ERcompared with the control group,there were no siginificant difference of mRNA and protein expression levels of ER a in Lead exposure group rats(P>0.05),but lead exposure groups rat amygdala ER ? mRNA and protein expression levels compared with the control group,were significantly down regulated(P<0.05).Conclusions1.Lead exposure can lead to increased blood lead content and lead content in the amygdala in the development period,both of which were positively correlated with the lead exposure time.The lead content in the amygdala increased with the rise of the level of blood lead and they had positive correlation.2.lead exposure can damage the structure of the amygdala in rats and affect the cognitive ability of rats.3.Lead exposure can down regulate the expression of amygdala ER? mRNA and protein levels,indicating that the ER? involved in regulation of cognitive ability. |
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