The Expression And DNA Methylation Of EphA5in The Brain Of Rat With Congenital Hypothyroidism

Objective:To establish the model of rat with congenital hypothyroidism, as well as to primarilyculture hippocampal neuron of fetal rat and further to intervene by group.Methods:1. Pregnant rats of G0(Gestational day, GD) were randomly divided into3groups:(1) the hypothyroid group, clean drinking water containing0.02%MMI was fed to thedams from G9until the pups were all executed,(2) the T4therapeutic group, cleandrinking water containing0.02%MMI was fed to the dams as same as the hypothyroidgroup, and the pups were treated with thyroxine by abdominal subcutaneous injectionseveryday from P7(Postnatal day, PD),(3) the control group, the dams were fed with cleandrinking water all the time.2. We observed the general biological characteristics of all the dams and pups, anddynamically monitored their weight change and the level of serum thyroid hormone aswell.3. The hippocampus, cerebral cortex and cerebellum of3groups were collected atP0,3,7,14and21individually. One part of the samples were stored in-80℃refrigeratorand reserved for the analysis of gene and protein. Another part were embedded withinparaffin and cut coronally for immunofluorescent staining.4. The fetal rats of hypothyroid and control group were obtained at G17and isolatedfor hippocampus after decapitation. Hippocampal neurons were primarily cultured with theserum free medium which is called neurobasal containing B27and glutaminate. Weobserved the morphological changes of hippocampal neurons under inverted phase contrast microscope and took the hippocampal neurons of7day for identification and puritycalculation by MAP-2.5. Hippocampal neurons of the hypothyroid group were divided into3groupsaccording to the medium:(1) T3group, the medium containing T3,(2) Azadc group, themedium containing Azadc,(3) Hypo-group, the serum free medium. Hippocampalneurons of the control group cultured during the same period were regarded as the controlgroup. Effects of different mediums on the growth of hippocampal neurons of thehypothyroid group were observed under the microscope. Activity of hippocampal neuronsof the above four groups were further determined by MTT colorimetric technique.Results:1. At G18, the weight of pregnant rats of the control group was significantly higherthan that of the hypothyroid group and the T4therapeutic group (P<0.05). The weight ofthe dams of3groups all dropped suddenly after the birth of the pups, then graduallyincreased during the whole location. But the acceleration speed of the weight of the controlgroup exceeded that of the hypothyroid group and the T4therapeutic group. The weight ofthe dams of the control group was significantly higher than that of the hypothyroid groupand the T4therapeutic group at P0,3,7,14and21(P<0.05). At the end of location, thelevel of serum FT3and FT4showed no difference between the hypothyroid dams and theT4therapeutic ones (P>0.05), but indicated marked difference in either the hypothyroidgroup or the T4therapeutic one and the control group (P<0.05).2. Compared with the control group, pups in the hypothyroid group and the T4therapeutic one appeared smaller size, sluggish, unresponsive, hair growth late and glossdecrease. In addition, the time points of opening eyes initially and completely in thehypothyroid group were both later than that of the T4therapeutic group and the controls.The birth weight of the pups in the control group was significantly higher than that of thehypothyroid group and the T4therapeutic group (P<0.05). Although the weight of the T4therapeutic group at P14demonstrated significantly higher than that of the hypothyroidgroup, it was markedly lower than the control group (P<0.05). Until P21, there was nodifference of the weight between the T4therapeutic group and the controls (P>0.05). Thelevel of serum FT3and FT4in the T4therapeutic pups showed markedly higher than thehypothyroid pups, however lower than the controls (P<0.05). Until P21, the level of serumFT3and FT4in the T4therapeutic pups reached that in the controls, and the level of either group was significantly higher than that of the hypothyroid pups.3. The observation of morphological changes of the hippocampal neurons undermicroscope showed that hippocampal neurons in the hypothyroid group appeared smallercell body, shorter neurite, the fewer number of projections, and lower degree of intensivenetwork formed by projections when compared with the controls at the same time.However, the purity of hippocampal neurons in both groups reached more than96%.Hippocampal neurons in the hypothyroid group presented longer neurite, the more numberof projections, higher degree of intensive network when intervened with different drugs.The analysis by MTT demonstrated that the activity of hippocampal neurons in eitherAzadc group or T3group was markedly higher than that in Hypo-group, but the above3groups were all significantly lower than the controls (P<0.05).Conclusions:The level of thyroid hormone and the general biological characteristics of both damsand pups indicate the successful production of model of congenital hypothyroid rat andtherapeutic rat. The observation of morphology and the identification and the activitydetermination of hippocampal neurons indicate the successful primary culture of normaland hypothyroid fetal rats and the successful grouping by drug intervention as well. Objective:To study the expressive characteristics of ephrin-A5/EphA5and several key moleculesin ephrin-A5/EphA5downstream signaling such as NMDAR-1（NR1）, PSD95andCaMKII in the brain of rat with congenital hypothyroidism. And to investigate therelationship between ephrin-A5/EphA5signal pathway and synaptogenesis disorder causedby thyroid hormone deficiency.Methods:1. The mRNA expression of ephrin-A5, EphA5, NR1, PSD95and CaMKII in the hippocampus, cerebral cortex and cerebellum of hypothyroid and therapeutic group and thecontrols at P0,3,7,14and21individually was measured by quantitative real time RT-PCR.And the protein expression was verified by Western Blot in these samples and furthermeasured by immunofluorescence.2. Hippocampal neurons of7day in the control group and the hypothyroid groupincluding T3group and Hypo-group were taken as the samples. Real time PCR andWestern Blot assay were used to measure the gene and protein expressive levels ofephrin-A5, EphA5, NR1, PSD95and CaMKII. Immunofuorescent staining was performedto assess the protein expressive level of the above five molecules.Results:1. In the brain of congenital hypothyroid rat, the gene and protein expressive levelsof ephrin-A5, EphA5, NR1, PSD95and CaMKII were all down regulated with remarkablespatial and temporal characteristics.(1) The results were obtained from comparison amongthe different empirical groups: from P0to P21, the gene and protein expressive levels ofephrin-A5, EphA5, NR1, PSD95and CaMKII in the hippocampus, cerebral cortex andcerebellum of hypothyroid group were all markedly lower than that of the controls(P<0.05), and the time point of the biggest decline was almost at P7except for NR1in thecerebellum; from P0to P7, there was no significant difference between the therapeutic andhypothyroid group in either the mRNA or protein expressive in the hippocampus, cerebralcortex and cerebellum (P>0.05), but the expressive level of the control group wassignificantly higher than that of the hypothyroid group and markedly lower than the controlgroup from P14to P21(P<0.05).(2) The results were obtained from comparison amongthe different brain tissues: the mRNA and protein levels of ephrin-A5, EphA5, NR1,PSD95and CaMKII were highest in the hippocampus, followed by the cerebral cortex andcerebellum the lowest in the hypothyroid group, the therapeutic group and the controlsfrom P0to P21; And the level of the biggest decline was in the hippocampus of thehypothyroid group.(3) The results were obtained from comparison among the differenttime points during the development: the time point when the expressive level ofephrin-A5、EphA5、NR1、PSD95、CaMKII in the hippocampus, cerebral cortex and cerebellum of the hypothyroid group markedly increased was postponed than that of thecontrols, and the time point of peak value delayed or vanished.2. In hippocampal neurons of primary culture, the expressive levels of ephrin-A5,EphA5, NR1, PSD95and CaMKII in Hypo-group were significantly lower than that in thecontrols (P<0.05). The expressive level of T3group was significantly higher than that ofHypo-group but still markedly lower than that of control group (P<0.05).Conclusions:Ephrin-A5/EphA5signal pathway would participate in the synaptogenesis disorder ofneuron in congenital hypothyroid rat. The down regulation of expressive levels ofephrin-A5、EphA5、NR1、PSD95、CaMKII cannot be entirely reversible by the replacementtherapy with thyroxine. Objective:To detect the level of DNA methylation of EphA5promoter in the hippocampus ofcongenital hypothyroid rat. And to investigate the relationship between DNA methylationand the expressive regulation of EphA5in the hippocampus of rat with congenitalhypothyroidism.Methods:1. Both the hippocampus at P7and hippocampal neurons of7day in the hypothyroidgroup and the controls were regarded as the samples.2. The level of DNA methylation of EphA5promoter CPG island in all the sampleswas detected by BSP cloning sequencing. And the activity of DNMT of all the samples wasdetected by kit.3. Hippocampal neurons of7day in the hypothyroid group including Azadc groupand Hypo-group were taken as the samples. The level of DNA methylation of EphA5promoter CPG island of hippocampal neurons was detected by BSP cloning sequencing. Real time PCR and Western Blot assay were used to measure the gene and proteinexpressive levels of EphA5at the same time.Results:1. The levels of DNA methylation of EphA5promoter CPG island in both thehippocampus and hippocampal neurons of the hypothyroid group were significantly higherthan that of the controls (t=10.15,13.74; p<0.001), which were both negatively correlatedwith mRNA expression of EphA5(r=-0.957,-0.831; p<0.05).2. The activities of DNMT in both the hippocampus and hippocampal neurons ofhypothyroid group were significantly higher than that of the controls (t=36.01,63.17;p<0.001), which were both negatively correlated with mRNA expression of EphA5(r=-0.998,-0.831; p<0.05).3. The level of DNA methylation of EphA5gene was markedly dropped (t=7.33,p<0.05) while the levels of mRNA and protein of EphA5were both significantly upregulated (t=7.07,6.42; p<0.05) in Azadc group when compared with Hypo-group.Conclusions:DNA methylation of EphA5gene would participate in the expressive regulation ofEphA5in the hippocampus of congenital hypothyroid rat. The expression of EphA5inhippocampal neuron of rat with congenital hypothyroidism could be up-regulated bydemethylation therapy with Azadc.