| Objective:1.To explore the changes of cognitive function of workers exposed to aluminum and its influencing factors,and to provide some reference measures for improving the cognitive function of workers.2.To investigate the changes of cognitive function and the expression of glutamate receptor protein in peripheral lymphocytes and the changes of low frequency amplitude ALFF in functional imaging in workers exposed to aluminum,and to explore the possibility of glutamate receptor and ALFF in peripheral blood lymphocytes as markers of cognitive function changes.3.To investigate the effects of aluminum exposure on synaptic plasticity and glutamate receptor expression in rats,so as to provide a basis for the study of the mechanism of cognitive impairment including learning and memory and future intervention.Methods:1.Population epidemiological survey and resting state functional imaging:1)Using the method of cluster sampling,254 workers exposed to high aluminum and 253 workers who were exposed to low aluminum in auxiliary work were selected as the subjects of the study,they were divided into high aluminum exposure group?later called high aluminum exposure group?and low aluminum exposure group?later called low aluminum exposure group?.The general data of workers were collected by employee health questionnaire,and the cognitive function of workers was evaluated by Montreal Cognitive Assessment scale.The plasma aluminum concentration of the exposed workers was detected by inductively coupled plasma mass spectrometry?ICP-MS?as an internal exposure index.The protein expressions of ionic glutamate receptor?NMDAR?and metabotropic glutamate receptor?mGluR1?in peripheral elbow vein plasma of workers were measured by enzyme linked immunosorbent assay?ElISA?.2)Based on the results of the previous population survey,27 workers in the exposure group?MoCA score<26?and 26 male workers in the non-exposure group?MoCA score?26?were scanned by resting state functional imaging?rs-fMRI?according to the MoCA score.The magnetic resonance images were obtained by3.0T high field intensity magnetic resonance scanner,the T1 weighted?T1WI?high resolution whole brain structure images were obtained by magnetization preparation fast gradient echo?MP-RAGE?pulse sequence,and the rs-fMRI data were obtained by single shot echo plane imaging?EPI?pulse sequence.DPARSF toolkit was used to analyze the low frequency amplitude of 0.01-0.08Hz band.2.Animal experimental study:40 healthy male SD rats were selected and fed adaptively in the Labor Health Animal Laboratory of Shanxi Medical University for one week,and then underwent lateral ventricular intubation.One week after operation,they were randomly divided into three groups according to their body weight:operation control group?only lateral ventricle intubation?,normal saline control group?injection of saline5?L?and maltol aluminum exposure group?injection of 40mM Al?mal?3 5?L?.They were exposed to the poison at 9:00 a.M.every two days for ten days in a row.After the exposure of Al?mal?3,the short-term memory ability of rats was detected by new object recognition test,and the field excitatory postsynaptic potential?fEPSP?in hippocampal CA1 region was recorded by electrophysiological technique,the number of dendritic spines in hippocampal neurons was observed by Golgi staining,the ultrastructural changes of neurons in hippocampal CA1 region were observed by transmission electron microscope,and the expression levels of mRNA of hippocampal mGluR1 and NMDAR subunits were detected by real-time fluorescence quantitative PCR.The protein expression of mGluR1and NMDAR subunits were detected by Western Blot.Results:1.Population epidemiological investigation:?1?Plasma aluminum level:?1?the average concentration of plasma aluminum in high aluminum exposed group was 39.58?24.49,60.17??g/L,while that in low aluminum exposure group was 17.98?6.42,37.03??g/L.The level of plasma aluminum in the high aluminum exposure group was higher than that in the low aluminum exposure group,and the difference was statistically significant?P<0.001?.?2?Analysis of general situation and comparison of cognitive function of the subjects:all subjects were male,the average age of the high aluminum exposure group was?41.53±7.70?years and the average length of service was?20.54±8.66?years.While the mean age of the low aluminum exposure group was?46.00±6.15?years and the mean length of service was?25.70±6.78?years.There were no statistically significant differences in the distribution of marital status,per capita monthly income,smoking and diabetes between the two groups?P>0.05?,but there were statistically significant differences in the distribution of age,working age,education level,alcohol consumption and hypertension between the two groups?P<0.05?.There was no significant difference in the scores of naming,attention,language,delayed recall and orientation between the two groups?P>0.05?,but there were significant differences in clock painting,total scores on visual space,abstraction and MoCA between the two groups?P<0.05?,and the score of the high aluminum exposure group were lower than that of low aluminum exposure group.?3?Analysis of the total score of MoCA scale and the influencing factors of each factor score:stepwise multiple linear regression and binary logistic regression analysis showed that plasma aluminum level,age,education level,drinking and total length of service were related to the total score or each factor score of MoCA.Age,total length of service and plasma aluminum level were the risk factors affecting the total score or each factor score of MoCA,and education level was the protective factor affecting the total score or each factor score of MoCA?P<0.05?,while the relationship between alcohol consumption and MoCA scores is not clear.?4?Analysis of factors influencing the cognitive performance:the unconditioned logistic regression analysis of influencing factors of cognitive impairment was conducted with the MoCA total score of 26??26 is considered normal,<26 is considered MCI possible?.The results showed that the risk factors for cognitive impairment were plasma aluminum exposure and age,while the protective factor was high education.?5?The expression of mGluR1 and NMDAR in the peripheral blood lymphocytes of the subjects:the expression of NMDAR1,NMDAR2A and NMDAR2B in peripheral blood lymphocytes in the high aluminum exposure group was lower than that in the low aluminum exposure group,while the expression of mGluR1 in the high aluminum exposure group was higher than that in the low aluminum exposure group,the differences were significant?P<0.05?.?6?Analysis of correlation between mGluR1 and NMDAR protein expression and plasma aluminum content,total score of MoCA and scores of various factors:Spearman rank correlation analysis showed that the scores of clock drawing,naming,delayed recall and orientation were not significantly correlated with the protein expression of mGluR1and NMDAR.The protein expression of NMDAR1 and NMDAR2A in NMDAR subunit was negatively correlated with blood aluminum content,while the expression of mGluR1protein was positively correlated with blood aluminum content?r=-0.281,-0.306,0.166,P<0.001?;There was no significant correlation between NMDAR2B protein expression and blood aluminum content,NMDAR1 and NMDAR2A protein expression was positively correlated with MoCA total score,visual space,attention,language and abstract score,NMDAR2B and mGluR1 protein expression was only correlated with abstract score,in which NMDAR2B protein expression was positively correlated with abstract score,while mGluR1 protein expression was negatively correlated with abstract score?r=0.098,-0.116,P<0.05?.2.Study on functional imaging of resting state:?1?The comparison of basic conditions of subjects:a total of 53 subjects were included in the study of the nuclear magnetic resonance?NMR?,including 27 subjects in the high aluminum exposure group and 26 subjects in the low aluminum exposure group,all of whom were males and were right-handed.The average age of the high aluminum exposure group was?46.70±5.42?years,and that of the low aluminum exposure group was?47.00±4.64?.There was no significant difference in age between the two groups,but the education years and MoCA scores of the low aluminum exposure group were higher than those of the high aluminum exposure group,while the blood aluminum level of the low aluminum exposure group was lower than that of the high aluminum exposure group,and the difference was statistically significant?P<0.05?.?2?The comparison of rs-fMRI ALFF results of subject:Compared with the low aluminum exposure group,the higher ALFF values in the high aluminum exposure group were mainly located in the bilateral superior frontal gyrus/auxiliary motor area and the left medial superior frontal gyrus,while the lower ALFF values in the high aluminum exposure group were mainly in the right cerebellum,left inferior temporal gyrus and left inferior parietal lobule than those in the low aluminum exposure group.?3?The correlation analysis between MoCA score and rs-fMRI ALFF in each brain region:in aluminum high exposure group,visual spatial execution score was positively correlated with rs-fMRI ALFF in infratemporal gyrus?r=0.402,P<0.05?,attention score was positively correlated with cerebellar rs-fMRI ALFF?r=0.395,P<0.05?,orientation score was positively correlated with rs-fMRI ALFF in medial superior frontal gyrus?r=0.421,P<0.05?.3.Animal experiments:?1?The basic conditions of rats:after the operation of the lateral ventricle,except for one rat who died,all the others recovered well.During the follow-up experiment,only aluminum-exposed 40mmol/L Al?mal?3 group had varying degrees of loss of appetite,range of activity,rough hair,depilation and other phenomena;other operation control group and saline control group had normal appetite and range of activity,and body weight increased with the increase of feeding time.?2?The results of new object recognition:there was no significant difference in exercise speed among the three groups.The NOI of the operation control group was?66.48±12.53%?,and that of the saline control group was?68.11±18.18%?,and there was no significant difference between the two groups.The NOI of the 40mmol/L maltol aluminum group was?51.25±15.09%?,which was significantly lower than that of the saline control group and the operation control group?P<0.05?.?3?The results of LTP in hippocampus of rats:the results of multivariate analysis of variance of repeated measurements showed that the average amplitude of normalized fEPSP in hippocampal CA1 increased after the administration of HFS,and the average amplitude of normalized fEPSP decreased after the exposure of Al?mal?3.There was an interaction between exposure and stimulation time in rats?F=17.852,P<0.05?.In the same treatment group,the average amplitude of normalized fEPSP decreased with the extension of the stimulus recording time?P<0.05?.In the different treatment groups at the same time,the average amplitude of normalized fEPSP decreased compared with the control group?P<0.05?.There was no significant difference in the average amplitude of normalized fEPSP at each time point between the operation control group and the saline control group.At the 0min,there was no difference in the average amplitude of normalized fEPSP in the hippocampal CA1 area in each group?P>0.05?.At 1min,the average amplitude of normalized fEPSP in the 40mM Al?mal?3 group was 34.9%lower than that in the normal saline control group.At 30min,the average amplitude of normalized fEPSP in the 40mM Al?mal?3 infection group was 41.0%lower than that in the saline control group.At 60min,the average amplitude of normalized fEPSP in the 40mM Al?mal?3 infection group was45.1%lower than that in the saline control group.?4?The results of the morphological observation in the hippocampal neurons of rats:after Golgi staining,it was found that in the operation control group and saline group,the neurons in the hippocampus of rats were rich in dendrites,full cell body,longer axon extension and more dendritic spines;after exposure to Al?mal?3,neurons atrophied,axon branches decreased,breakage and bead-like changes appeared,and the number of dendritic spines decreased.The density of dendritic spines in CA1 area of hippocampus was further observed under oil microscope.The density of dendritic spines in operation control group was slightly higher than that in normal saline control group,but the difference was not statistically significant?P>0.05?.The density of dendritic spines in aluminum exposed group?40mM Al?mal?3?was lower than that in normal saline control group and operation control group,and the difference was statistically significant?P<0.05?.The results of transmission electron microscope observation showed that the structure of mitochondria in the hippocampal neurons of the two control groups was intact,the synapses were dense and well shaped,the postsynaptic dense bands were clearly visible,the synaptic vesicles were abundant in the axons,and the endoplasmic reticulum and ribosomes were abundant in the dendrites.In the Al?mal?3 exposed group,the mitochondrial crest was broken,and there was obvious swelling,the ribosomes and endoplasmic reticulum in the dendrites decreased,the synaptic structure was destroyed,the vesicles in the anterior membrane decreased,the postsynaptic densification became thinner,and the axons also showed different degrees of edema.?5?The mRNA and protein expression of mGluR1 and NMDAR subunits in hippocampal tissues of rats:there was no statistically significant difference in mRNA expression of mGluR1 and NMDAR subunits between the operation control group and the saline control group?P>0.05?.Compared with the saline control group,the expression of mGluR1 mRNA in the 40mM Al?mal?3 group was increased,but the expression of NMDAR1 mRNA and NMDAR2A mRNA were decreased.The expression of NMDAR2B mRNA was not significantly different from that of saline group?P>0.05?.The protein results showed that there was no statistical difference in the expression of mGluR1 and NMDAR subunits between the operation control group and the saline control group?P>0.05?.Compared with the saline control group,the changes of the protein expression of mGluR1,NMDAR1,NMDAR2A and NMDAR2B in 40mM Al?mal?3 group were consistent with those of mRNA expression,and the differences were statistically significant.?P<0.05?.Conclusion:1.The level of aluminum in plasma of long time occupational aluminum exposed workers was higher,and their cognitive function was impaired.2.The expression of NMDAR1,NMDAR2A and mGluR1 proteins in human peripheral blood lymphocytes was correlated with plasma aluminum content and the scores of MoCA and its factors.In the workers with relatively high plasma aluminum levels and relatively poor cognitive function,it was observed that the ALFF indexes related to memory,emotion and emotion decreased,and the spontaneous activity of neurons decreased.3.The expression of mGluR1 in hippocampus of rats exposed to Al?mal?3 increased,while the expression of NMDAR1 and NMDAR2A decreased,which was consistent with the changes of peripheral blood levels in workers exposed to aluminum,suggesting that the mechanism of the effect of aluminum on cognitive function may be related to the effect of aluminum on the expression of glutamate receptor. |
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