Study The Intelligence Of School-age Children In Coal-burning-borne Endemic Fluorosis Areas And The Damage And Prevention Of Learning And Memory Ability In Offspring Rats With Chronic Fluorosis

Objective：Endemic fluorosis is a disease distributed widely in the world, andexerts toxiceffects on many tissues and organs of human body, including centralnervous system (CNS), lead to children’s mental retardation in endemic area.However, the molecular mechanism of developing brain injury induced by chronicfluorosis is still not clear at present. We conducted the survey in order to understandthe intellectual development of school-age children after a certain time ofcomprehensive management, the aim of the study is to investigate the changes ofNMDAR、calcium ion pathway and oxidative stress in the animal models and cellmodels of chronic fluorosis, and further reveal the role of excitatory neurotoxicity inthe mechanism of brain injury caused by chronic fluorosis. At the same time,application of antioxidant vitamin E to find effective measures to prevent chronicfluorosis.Methods： Children aged8-12who lived in coal-burning-borne endemicfluorosis areas in Bijie City of Guizhou Province were selected and divided into twogroups according to the duration of comprehensive treatments given: long treatmentgroup (furnace stove was changed and comprehensive control measure of healtheducation was carried out for more than3years) and short treatment group (stoveswere improved and health education time <1year). The children who lived in anon-fluorosis area were selected as controls. Dental fluorosis was diagnosed by themethod of Dean; urinary fluoride was analyzed by the method of Fluoride-ion selective electrode; and the intelligence quotient (IQ) was measured by Raven’sStandard Progressive Matrices Test.This experiment replicated the rat models with chronic fluorosis by feeding thedrinking water containing sodium fluoride (NaF) and vitamin E gavage treatment.Five months after established the animal model, the rats were mated in2:1proportionof female:male in different groups, postnatal1、7、14、21、28d offspring rats wereexperimented, respectively.The body weight and neurobehavioral development indexsin offspring were observed; Spatial learning and memory ability were evaluated byMorris Water Maze test; the changes of neuropathology by HE and Nissl stainingwere observed under microscopy; oxidative stress indicators were measured byrelated kits;The distribution of Neu N、NMDAR（NR1、NR2A、NR2B、NR3A）、CaMK II in brains were detected by immunohistochemistry. The expressions ofNMDAR subunits at protein and mRNA levels were measured by Western-blottingand Real-time PCR, respectively. Primary neuronal cell cultured and treated withfluoride in vitro.The neural cell apoptosis rate in brain tissue of offspring and in vitroculture cells were detected by flow cytometry;The expressions of NMDAR subunitsat protein and mRNA levels were measured by Western-blotting and Real-time PCR,respectively.Results: The results showed that the dental fluorosis inspection rate and thefluoride content in urine of children in fluorosis endemic area were significantlyhigher than the control group, And long–time treatment group detection rate washigher than the short-time treatment group. Intelligence survey showed that children’sIQ above average proportion decreased in flurosis areas, IQ distribution negativelycorrelated with urinary fluoride content. IQ level in long-time treatment andshort-time treatment group showed were no significant differences in the overall andgender distribution. However, the low age group children’s IQ medium aboveproportion in the long time comprehensive treatment group has increased.Resluts of animal experiments showed that the birth weight of offspring ratswere no significant difference, but postnatal28d the weight of offspring in fluorosis group were significantly lower than the control group and Vit E intervention group; incontrast to control group,the time that completed the surface righting reflexand the airrighting reflex were delayed in the offspring in fluorosis group and antagonistic group,the average escape latency and exploration platform were decreased in offspring offluorosis group. A number of neuropathological changes, including the nucleusproportion increase, enhanced dyeing, decreased nissl body, Reduced expression ofneurons core antigen (Neu N) were found in offspring brain with chronic fluorosis;the cell apoptosis rate of brain tissue increased; SOD activity decreased, MDAcontent increased, showed an increased trend of NO and NOS. The indicators in Vit Eintervention group were no significant difference compared with control group.Compared with the protein expression in newborn rats, in Control group, NR1proteinexpression in postnatal21d elevated to the peak, then declined gradually;Expression of NR2A protein increased significantly in postnatal14d, and maintainedto postnatal28d; NR2B subunits protein expression increased obviously in postnatal28d; NR3A subunits had the highly expression in postnatal14d of offspring rats;The expression of Phospho-NR2B increased in14d rats, then remained stability to28d rats; CaMK II protein expression increased significantly in postnatal14d, thendeclined slowly. In fluorosis group, expression of NR1subunit increased in7doffspring rats, then gradually decreased to a newborn level; NR2A subunit proteinhad highest expression in postnatal28d; expression of NR2B in offspring rats of14dincreased significantly; expression of NR3A had no statistical significance;expression of Phospho-NR2B increased in postnatal14d rats and increasedsignificantly in21d and28d; expression of CaMK II protein had no significantchange. In Vit E treatment group, expression of NR1subunit peak appeared in14drats, until to postnatal28d; the expression peak of NR2A、NR2B、NR3A andphospho-NR2B subunit appeared in postnatal21d、14d、7d、14d, respectively;expression of CaMK II protein in Vit E treatment group increased in7d andmaintained to postnatal28d. Compared with control and Vit E treatment group, theexpression of NR1、 NR2B and phospho-NR2B protein increased significantly in fluorosis group, CaMK II、NR3A protein level declined, and expression of NR2Aprotein had no obvious change. The mRNA levels of NMDAr each subunitincreased with the growth and development of offspring rats, the NR1and NR2bmRNA expression levels had obviously increased in21d rats, then expression levelsdecreased in28d offspring rats in fluorosis group, NR3a mRNA level declined; butNR3a mRNAlevel increased in Vit E intervention group.In this experiment, we successfully cultured primary nerve cell and treated withfluoride in vitro, the apoptosis rate and the fluorescence staining of intracellular ROSand Ca2+enhancement with the fluoride treat time increased; the expression ofNR1、NR2B、phospho-NR2B protein increased, NR2Aexpression had no significantchange; the expression of NR3A and CaMK II decreased. The results of Real-timePCR showed that the mRNA expression of NMDAR each subunit had no changedobviously in low fluoride treat group; the mRNA level of NR2B increased and NR3Adecreased in high fluoride treat group. The apoptosis rate and intracellular ROSfluorescence intensity decreased obviously after pretreatment by antioxidants Vit E,Ca2+fluorescence intensity had reduced.Conclusions: The results in our study showed,(1) the fluorosis situation ofschool-age children remains severe in endemic fluorosis areas. however, the children’sdental fluorosis inspection rate and the fluoride content in urine decreased, low agechildren intelligence improved significantly after a long time comprehensivetreatment.(2) Chronic fluorosis has a harmful effects on the offspring growth anddevelopment and differentiation of the nervous system.(3) Fluorine accumulation cancause oxidative stress levels in the brain tissue, reactive oxygen intermediates andreactive nitrogen intermediate and lipid peroxidation products increases, theantioxidant enzyme activity decreased.(4) Brain tissue fluorosis may activate NMDAreceptor function, expression of NR1, NR2B subunits、 phospho-NR2B levelsincreased; NR3A subunits expression decreased; Calcium ion channel opened,intracellular calcium ion concentration increased, cause cells to produce excitatory toxic damage, apoptosis necrosis rate increased, thus caused damage to the learningand memory and other functions.