| Objective:Maternal hypothyroidism affects the migration,differentiation and maturation of neurons,the growth of dendrites and axons,and the formation of synapses and myelination in offspring brain.Offsprings showed mental retardation,decreased verbal memory,even cretinism.In the 60s of last century,it was generally believed that thyroid hormone was not able to pass the placenta,and the related research on thyroid hormone affecting brain development was mainly concentrated in the postnatal stage.With the medical screening,molecular biological technology progress,people found that the fetus thyroid function has not been established had thyroid hormone receptor expression in their brain tissue and we can detect a certain amount of thyroid hormone in their serum.It shows that the maternal thyroid hormone can reach the fetus through the placenta.Before the establishment of fetal thyroid function,lacking of thyroid hormone can also lead to a decrease in intelligence.The development of mammalian brain is carried out in a certain time and space sequence,and the regulation of thyroid hormones has time and space specificity.That is,thyroid hormone has different functions on different tissues and different cells,and it also plays different roles in regulating the same tissue or cell in different stages.Whether there is a difference in the effect of thyroid hormone deficiency on the mental and neurodevelopment of offspring at different stages of brain development is still unclear.The perinatal thyroid hormone overdose leads to the acceleration of the neuron differentiation in the offspring.Gestational transient thyrotoxicosis is easy to occur during early pregnancy.Gestational transient thyrotoxicosis can affect the outcome of pregnancy.There is no related report on the effect of offspring brain development.The thyroid hormone regulates the target gene transcription through the nuclear receptor pathway and affects the expression of the neuron development related factors.It can form a heterodimer with a variety of nuclear receptors,and recruit transcriptional activation or transcriptional suppressor to play a different transcriptional regulation.The researchers screened the thyroid hormone target gene for many times and mainly focused on the offspring after birth.However,there is still a gap in the study of gene expression changes caused by maternal thyroid hormone deficiency at the early stage of brain development.In this study,we established maternal thyroid hormone deficiency model at different stages of brain development to explore the difference of thyroid hormone deficiency in different stages on offspring's neuropsychological development.We investigated the effect of maternal excess or deficient thyroid hormone in the early pregnancy on the mental and neurodevelopment of offsprings.We screened of thyroid hormone target gene in the early stage of brain development,to provides new clues for the mechanism of thyroid hormone affecting the early brain development of the offspring.Methods:Part 1.1.0.025%methimazole(MMI)of drinking water and 1.5?g/100g body weight per day levothyroxine(L-T4)intervention was used to establish gestational day 10(GD10)-GD17(equivalent of the human early brain development stage),GD17-postnatal day 0(PNDO)(equivalent of the human second brain development stage),and PNDO-PND 10(equivalent of the human third brain development stage)maternal thyroid hormone deficiency rat model.The normal control group was given normal drinking water.2.The blood samples were collected after model establishment and on the second day of treatment intervention.Serum total thyroxine(TT4)and thyroid stimulating hormone(TSH)levels in pregnant rats were detected by ELISA.3.Offspring rat blood was taken on PND 10,and serum TT4 and TSH was detected by ELISA.The TT4 in offspring brain tissue was detected by ELISA method after homogenate.4.On PND 10,righting reflex and tentacle induced forelimb placement test were carried out to detect the somatosensory and motor cortex function of the offspring.The synaptic plasticity and synaptic transmission in the hippocampal CA1-CA3 region of the offspring were detected by long-term potentiation.5.On PND40,open field experiments,the elevated plus maze,zero maze,rotation acceleration test,Morris' water maze and forced swiMMIng test were used to detect offspring's activity,anxiety,depression,exercise ability and learning and memory ability in turn.Part 2.6.0.025%MMI of drinking water and 1.5?g/100g body weight per day L-T4 intervention to was used to establish GD10-GD17 maternal thyroid hormone deficiency rat model.1?g/100g body weight per day levothyroxine(L-T4)was used to establish maternal thyroid hormone excess rat model.Normal control group were given normal drinking water.7.The blood samples were collected after model establishment and on the second day of treatment intervention.Serum total thyroxine(TT4)and thyroid stimulating hormone(TSH)levels in pregnant rats were detected by ELISA.8.On PND10,TT4 was detected by Roche electrochemiluminescence(Roche).TT4 in brain tissue was detected by ELISA method after homogenate.9.On PND40,MWM and fear condition experiment was used to detect the learning and memory ability of the hippocampus and cerebral cortex of offspring.10.On PND40,adult offsprings were tested for long time potentiation to detect the synaptic plasticity and synaptic transmission in the hippocampal CA1-CA3 region.11.On PND40,argentaffin staining was carried in brain paraffin section to observe the axon of neurons.12.RT-PCR were used to detecte thyroid hormone responsive genes,neurotrophic factor and axon guidance factor expression in cerebral cortex and hippocampus of rats at adult PND40.13.the Illumina HiSeqTM4000 platform was used to sequence the mRNA expression profiles of GD17 day fetal cerebral cortex,and analyze the gene differential expression induced by maternal thyroid hormone deficiency in early brain development.14.Nissl staining was used to observe the morphological and structural development of the cerebral cortex of GD17 fetus.Results:Part one:1.After 0.025%MMI intervention,the serum TT4 levels were significantly decreased in all groups compared with those in the CN group(p<0.05).The level of TSH in serum was significantly higher than that CN group(p<0.05).The serum levels of TT4 and TSH were detected in each group after treatment.There was no difference in TT4 and TSH between the experiment group and the control group(p>0.05).2.The number of childbirth in H1 group was less than that of CN group(p<0.05).There was no difference in the number of H2 and H3 offspring compared with CN group.In H1,H2 group,the weight of offspings in PND5 was lower than that in CN group(p<0.001).Compared with CN group,the weight of H3 offspings decreased in PND5 and PND10(p<0.001).3.At PND 10,there was no difference in the serum TT4 between four groups(p>0.05),and there was no difference in the serum TSH between four groups(p>0.05).There was no significant difference in TT4 level in the PND 10 brain tissue of the offspring of four groups,p=0.074.4.The accuracy of tentacle reflex in H1 group was lower than CN group(p<0.05)after birth(p<0.05).The PND5 righting reflex time of H1 and H3 group was longer than that of the normal group(p<0.05).5.H1 group,the percentage of field excitatory postsynaptic potential amplitude and the percentage of increase in slope of excitatory postsynaptic potential was lower than that in CN group in PND10 after high frequency tetanus stimulation,p<0.05.T the percentage of field excitatory postsynaptic potential amplitude after high frequency tetanic stimulation in H2 and H3 groups was lower than CN control group(p<0.05).6.The results of open field test showed that compared with CN group,the number of standing times in the H3 group in 30min decreased(p<0.05).The results of the elevated plus maze showed that standing frequency,the exploration behavior were decreased in the H1 group(p<0.05).The results of zero maze showed that the grooming times and the number of open entries decreased in Hl group(p<0.05).7.MWM showed that at DAY2,the escape latency of H1,H2 and H3 groups was prolonged(p<0.05).The second part:1.The serum TT4 in Hypo group was significantly lower than CON group,and the TSH was significantly higher than CON group at GD10(p<0.05).The serum TT4 in Hyper group was significantly higher than CON group,and the level of TSH was significantly lower than CON group at GD10(p<0.05).The sermm levels of TT4 and TSH were recovered to normal level(p>0.05)after L-T4 treatment in Hypo group.2.There was no significant difference in serum TT4 of offspring rats at PND 10(p>0.05).There was no significant difference in TT4 level in the brain of each group at PND10(p>0.05).3.In MWM,the lantency of Hypo and Hyper groups was longer than CON group(p<0.05)at the second and third days of training.4.Fear conditioning memory test showed that on the second day of the associated test,the frequency of freezing of Hypo group was lower than CON group and Hyper group in 5min(p<0.05),but the total freezing duration was higher(p<0.05).5.LTP showed that the percentage of field excitatory postsynaptic potential amplitude and the percentage of increase in slope of excitatory postsynaptic potential in Hypo and Hyper group was lower CN group after high frequency tetanus stimulation(p<0.05).6.Silver staining of axons in P40 neurons showed that neurofibrils and axons in Hypo group and Hyper group were fewer,and the axonal development was abnormal.7.There was significant difference in related thyroid hormone response genes,neurotrophic factors,nerve guiding factors and GTPase family factors in Hypo and Hyper group compared with CON.The third part:1.At GD17,the cerebral cortex of the offspring of hypothyroidism mother was lagged,the neurons were arranged in disorder,and the structure of each layer of the developing cortex was not obvious.The thickness of cerebral cortex and cortical plate of hypo group in early pregnancy was significantly lower than that of normal rats(p<0.001 and p<0.001).2.In Hypo group,the expression of hairless mRNA was lower than CON group in the cerebral cortex and the expression of MBP,and CamKIV mRNA was lower than CON group in the hippocampus on PND 10.Conclusion:The maternal thyroid hormone deficiency at different stages of brain development has different effects on the development of the offspring's mental and neurologic development.In the early stage of brain development,maternal thyroid hormone deficiency and excess affect the learning and memory ability related to hippocampus and cerebral cortex in the adult rats,and cause the dysplastic axonal development.The maternal thyroid hormone in the early stage of brain development may affect the development of the offspring's brain by affecting the expression of related neurotrophic factors,nerve guiding factors and GTPase family factors.The deficiency of maternal thyroid hormone in the early stage of brain development causes the differential expression of gene in the GD17 cerebral cortex and lead to the retardation of morphogenesis in the cerebral cortex of the offspring. |
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