Mechanism Study On Effects Of Mild Iodine Deficiency And Iodine Supplementation During Pregnancy And Lactation On The Cerebellar Motor Coordination In Rat Offspring

Objective:Iodine,as an essential component for thyroid hormone?TH?synthesis,is recognized as an important micronutrient for fetal growth and development.Iodine deficiency?ID?can result in neurodevelopment impairments,motor dysfunction,and cognitive function defection.Pregnant and lactating women are still at risk for mild deficiency in many countries,including developed nations.Mild ID is regarded as the most common cause of maternal and neonatal hypothyroxinemia.Hypothyroxinemia means abnormal TH level,characterized by the low level of thyroxine?FT₄?and unchanged level of triiodothyronine?T₃?and thyroid stimulating hormone?TSH?.It is generally considered that iodine supplementation is beneficial for pregnant women in severe ID.However,there is no definitive evidence of iodine supplementation during pregnancy in mild ID areas.Some epidemiological studies suggest that iodine supplementation to pregnant women may decrease the risk of cognitive and behavior developmental damage in the offspring.There are also some researches indicating that iodine supplementation is related to negative effects on children neurodevelopment.So it is still uncertain whether iodine supplementation to mild ID mother in pregnancy and lactation is useful for the offspring.TH,synthesized by iodine,is an essential hormone for normal cerebellum function.Body balance needs cerebellum whose neural pathway is concerned with motor coordination.Purkinje cell is the sole-output neuron from the cerebellar cortex.Harmful impact on mice motor coordination and motor behaviors can be exerted by morphological impairments of purkinje cell such as shorter length of dendrites,less branches at the same distance from soma and changes of purkinje cell ultrastructure.Motor coordination is believed to be under the control of slow excitatory postsynaptic current?sEPSC?,and the generation of sEPSC relies on metabotropic glutamate receptor type 1?mGluR1?signaling cascade.It is still unknown whether there is influence of maternal mild iodine deficiency and iodine supplementation on the morphology of purkinje cell and complexity of dendritic trees in rat offspring.The effect on motor coordination and mGluR1 signaling pathway remains to be elucidated.Therefore,mild iodine deficiency and iodine supplementation model was established by providing female rats with ID diet and deionized water containing different amount of potassium iodide to study whether maternal mild iodine deficiency and iodine supplementation would have an impact on the morphology of dendritic trees and the ultrastructure of purkinje cell,so that the motor coordination of pups would be changed.In this way,we can illustrate the effects of maternal mild iodine deficiency and iodine supplementation on the cerebellar development and motor coordination in rat offspring and its underlying mechanisms from motor behavior,morphology and molecular biology.Methods:1.Animals.Sixty female Wistar rats?130–150 g?were randomly assigned into four groups according to their weight:control group,mild ID group?MID?,Iodine supplementation group?MID+I?and severe ID group?SID?.ID diet(iodine content:about 60 ng/g,measured by As³⁺-Ce⁴⁺catalytic spectrophotometry)and deionized water supplemented with potassium iodide?KI?were provided for animals.Iodine intake of the female rats was 7.0,3.0,3.0 and 1.5?g/day in four groups respectively.Three months later,female rats were then mated with normal male rats??/?=2:1?.From gestational day 0?GD0?to postnatal day 21?PN21?,the pregnant rats of the control,MID and SID group were fed the same as before,but the iodine intake of MID+I group pregnant rats was upregulated to 7.0?g/day.2.Determination of TH and TSH.After ether anesthesia,blood samples were obtained from pups in every group on postnatal day 14?PN14?and PN21.At GD0,blood was obtained from dam rats.FT₄,FT₃ and TSH were measured by supersensitive chemiluminescence immunoassay.3.Determination of urinary iodine concentration.Urine samples were collected with metabolic cages for 24 h in early,middle,and late pregnancy of dam rats.The urinary iodine concentration?UIC?was measured by As³⁺-Ce⁴⁺catalytic spectrophotometry method.4.Behavioral tests.At PN14,The fore paws of each pup were suspended on a horizontal steel wire.The latency before falling from the wire was recorded.At PN21,we carried out rota-rod to record the latency of pups falling from the rota-rod.At PN21,balance beam test was carried out to record the time pups taken to traverse the beam.5.Golgi-Cox staining.The cerebella of pups were removed to perform Golgi–Cox staining at PN14 and PN21.Microscope was used to study the morphological characteristics of cerebellar Purkinje dendritic trees.The total length of dendritic arborization was quantified by Image J software.Sholl analysis was used to reveal the dendritic branching pattern complexity.6.Transmission electron microscope?TEM?.TEM analysis was performed to study ultrastructural changes in Purkinje cells.Pups at PN14 were anesthetized with ether.Cerebella were removed as quickly as possible and fixed by 2%glutaraldehyde in 0.1 M phosphate buffer.7.Western blotting.Pups were deeply anesthetized and euthanized at PN14 and PN21.Cerebella were removed from the skull and washed in an ice-cold artificial cerebrospinal fluid.The amount of G?q,m Gl UR1,PLD1 and TRPC3 was determined by western blotting.Each band was visualized with the Bioanalytical Imaging System.The relative density of the blot was quantified via ImageJ.8.Immunofluorescence.Pups were anesthetized and perfused with 4%paraformaldehyde at PN14 and PN21.Then the fixed cerebellum was embedded into paraffin and sectioned into 4?m thick sagittal sections.The images were obtained from fluorescence microscopy.The fluoresence intensity of mGluR1 and TRPC3 was analyzed.9.Statistics.Statistical analysis was performed using SPSS software,version17.0.Data was reported as mean±standard error of the mean?SEM?.One-way analysis of variance followed by Student-Newman-Keuls test was used to compare difference between four groups.A P value of<0.05 was considered statistically significant.Results:1.Alterations in serum TH and TSH level.There was no significant difference of FT₃ and TSH level in control,MID and MID+I group at GD0,PN14 and PN21?P>0.05?.Serum FT₄ level of dams at GD0 and pups at PN14 and PN21 in MID group was significantly lower than control group?P<0.05?.At GD0,FT₄ level of MID+I group was significantly lower than control?P<0.05?,but was not significantly different from MID group?P>0.05?.At PN14 and PN21,there was no significant difference in serum FT₄ level between MID+I group and the control?P>0.05?.But compared with MID group,FT₄ level significantly increased in MID+I group?P<0.05?.In SID group,serum FT₃ and FT₄ level were significantly lower than control and MID group?P<0.05?,and TSH level was significantly higher than control and MID group at any time point?P<0.05?.2.Alterations in UIC level.Compared with the control,there was a significant reduction in the UIC of MID group at early,middle and late pregnancy?P<0.05?.At early pregnancy,UIC level of MID+I group was significantly lower than control group?P<0.05?,but was not significantly different from MID group?P>0.05?.At middle and late pregnancy,there was no significant difference between MID+I and control group?P>0.05?,but UIC level of MID+I group showed significantly higher than MID group?P<0.05?.Compared with the control and MID group,the UIC level of SID group significantly decreased at all time points?P<0.05?.3.Behavioral alterations in MID and iodine supplementation group.In wire grip test at PN14,balance beam test and rota-rod test at PN21,the performance of MID pups was significantly inferior to control?P<0.05?.There was no significant difference between MID+I and control group?P>0.05?.Compared with MID group pups,the MID+I group pups performed significantly better?P<0.05?.SID group pups performed more poorly than MID and control group?P<0.05?.4.Morphological changes in dendritic arborization of Purkinje cells in MID and iodine supplementation group.Golgi-cox staining revealed the total length and complexity of dendritic arborization were significantly lower in MID group compared with the control at PN14 and PN21?P<0.05?.There was no significant difference of the total length between MID+I group and the control Purkinje cell dendritic trees?P>0.05?.But there was a significant increase of the total length and dendritic branching complexity in MID+I group compared with MID group?P<0.05?.In SID group,it appeared shorter length in dendritic trees of Purkinje cell than the control and MID group at both time points?P<0.05?.5.Ultrastructure changes of purkinje cells in MID and iodine supplementation group.For MID group,mild abnormal changes could be found in Purkinje cells at PN14.Electron micrographs of Purkinje cells demonstrated invagination of nuclear envelopes.Fracture and disappearance of ridges can be observed in part of mitochondria.In MID+I group,they showed normal cell bodies and cell nucleus.SID group revealed severe impairments of ultrastructure.Invagination of nuclear envelopes and shrinkage of nucleus could be seen and the margin of nuclear envelopes was toothed.We also found the fractured ridges of mitochondria and foreign matter in mitochondria.Dilated smooth endoplasmic reticulum was observed.6.MID and iodine supplementation affected the expression of proteins related to motor coordination.Our study showed there was a reduction in the expression of mGluR1,G?q,PLD1 and TRPC3 in MID group in comparison to the control at PN14 and PN21.There was no difference between MID+I and the control group in the expression of the proteins.We also found that the expression of the proteins increased in MID+I group in comparison to MID group.Compared with the control and MID group,there was a reduction in the expression of proteins in SID group.Conclusion:1.Maternal mild iodine deficiency may damage the morphology of dendritic arborization and ultrastructure of purkinje cell.2.Maternal mild iodine deficiency may damage the motor coordination via inhibiting the mGluR1 pathway.3.Iodine supplementation during early pregnancy may improve the impairments of purkinje cell and motor coordination caused by mild iodine deficiency.