The Effects Of Thyroid Hormone On Connexin Expression In The Cerebral Cortex Of Neonatal Murids

BackgroundCongenital hypothyroidism (CH), due to insufficient secretion of thyroid hormone (TH) result from variety of reasons, is a common pediatric endocrine disease. It is also a main cause of childhood mental retarded and draws more and more attention from endocrine pediatrician. With the development of molecular biology, physiology of TH has been better understood. TH binds its receptor (TH receptor, TR) in the nucleus, which plays a role on regulation the target gene transcriptional level by recognition the thyroid hormone response elements (TREs) in the promoters. However, the candidate genes which be regulated by TH and cause mental retardation in children with CH are still unclear. The signaling proteins and related proteins have effect on the information transfer and the various metabolic activity, and then on nerve cell growth and differentiation. They also take part in the mechanisms of sensory, motor, learning, memory and other activities. Hence, study the effect of TH on the signaling proteins and its related proteins may help us to better understand the effect of TH on brain development and the mechanism of CH.The classic information transfer in nervous system is the neurotransmitter transmittion throught the synapses, or the chemical synapses. Previous studies showed that TH had effect on the synthesis and degradation of neurotransmitters, and on the expression and function of their receptors. Recently, studies found several several non-classic information transfer pathway in the nervous system, such as gap junction (GJ) communication (GJC) signal transduction systems, which play important role on some nervous activities.GJ is the morphological substrate of one type of electrical synapse and mediates the GJC among adjacent cells. The key component of GJ is connexin (Cx). GJ is composed of a pair of connexon that includes six Cx proteins and make a hemichannel with a diameter of about 1.5 nm. There is abundant GJ in the nervous system, including at least 12 types of Cx. Cx43 is the predominant type and accounts for 90-95% of the total Cx protein. Moreover, types of Cx on different nerve cells are different. Cx32 is mainly in oligodendrocytes while Cx43 is mainly in astrocytes. Recently, Cx45 is also found in the central nervous system.Studies have shown that astrocytes interconnected as a syncytium through GJ and GJ may also constitute a complex of neurons-glial cells-neurons network. GJ allow the transfer of small molecules from cell to adjust cell, also information communication direct. Moreover, some Cx did not form a GJ with the adjacent cells, forming a direct channel for material and information exchange from the extracellular environments. Efficient intercellular communication at GJ is essential for normal electro-mechanical function of the brain. Compared with synapse, GJC has direct, fast and bidirectional characteristics. GJ has an important role on stability of the intercellular and extracellular environments, on nerve cell proliferation, differentiation, and development, also the signal transduction in brain. Several diseases have been associated with the disorder of GJ. Several studies showed that TH may affect the Cx43 expression on the myocardial and Sertoli cells, although the results are still controversial. However, no similar study about the effect of TH on the Cx in central nervous system was reported.Here, we established a CH model firstly, and measured the total levels Cx43, Cx32 and Cx45 on cerebral cortex in this model. Then, cultuered in the astrocytes by different concentration of T3 and measured the levels of these Cxs.ObjectivesTo investigate the effect of TH on Cx3 expression by measuring the Cx43, Cx32 and Cx45 expression in cerebral cortex in mice CH model, and these Cx in the astrocytes cultuered by different concentration of T3.Mice CH Model1 Materials and Methods1.1 Animal ModelHealth adult C57BL/6J mice mated together. They were divided into CH group and control group according to the different intervention. The control group is definited as feeding of clean drinking water. The CH group is definted as feeding 0.03% thiamazole contained water from the 10th day after mate to 7th day after the offsprings'birth. The offsprings were sacrificed on day 1,7,14 and 21. Saline perfusion was performed and the cerebral cortex was collected.1.2 Parameter MeasurementsThe levels of Cxs mRNA were measured by real time RT-PCR. Total RNA was extracted using AxyPrep Mutilsoure Total RNA Miniprep Kit. Self-designed primers and SYBR PrimeScriptTM PCR Kit were used real time RT-PCR. Two-step method was used for Cx43, Cx32, and Cx45. ACt was presented for target gene mRNA levels.The levels of Cx protein were measured by Western blot. Proteins were extracted by RIPA and the concentration was measured by BCA Protein Assay Kit. A 50μg total protein was used for SDS-PAGE electrophoresis and transferred PVDF. Then blocked, combined with first and second antibodies, and ECL radioautography in sequence. Finally, scan the radiation autoradiography strip and analysis the protein bands with gray-scale. The Cx/β-actin ratio was calculated as the relative levels of Cx. Tissue was fixed in 10% neutral formalin and embeded in paraffin. "EnvisionTM two-step" method was used for immunohistochemistry.2 ResultsIn control group, the levels of Cx43 expression on the cerebral cortex increased after birth and reach the peak at day 7, while the levels of Cx43 increased slowly in CH group. Commpred with the control group, the Cx43 mRNA levels in CH group were lower on day 1,7 and 14 without significant difference. Moreover, the Cx43 protein in CH group was lower than these in controls with a marginal difference on day 1 and a significant difference on day 7. Immunohistochemistry showed Cx43 protein was abundant on the cerebral cortex in both groups, including the body of nerve cells and nerve fibers.The levels of Cx32 were low on the cerebral cortex in the control and CH goups, and decreased after birth. Compared with the controls, the levels of Cx32 mRNA were significantly higher in the CH group on day 7,14 and 21, and the Cx32 protein increased with a significant difference on day 7 and a marginal difference on day 14. Immunohistochemistry showed that the expression of Cx32 was significantly less than the Cx43 on the cerebral cortex, and some nerve cells and few nerve fibers were positive.The levels of Cx45 were less than Cx43 and higher than Cx32, and decreased after birth. Both mRNA and protein of Cx45 had no significant difference between the two groups. Immunohistochemistry showed that Cx45 expression is similar to the distribution Cx43, and most body of nerve cells and nerve fibers was positiveCell Culture1 Materials and Methods1.1 Medium PreparationLow TH fetal bovine serum (activated carbon adsorbed FBS), common fetal bovine and serum T3 were purchased. DMEM solution with 5 nmol/L of T3 was prepared. A variety of DMEM culture mediums were prepared as following and TH were measured.MediumⅠ, common DMEM+ common FBS (TT3:5.4 nmol/L, FT3:22.0 pmol/L).MediumⅡ, common DMEM+ 15% low TH FBS (TT3:0.6 nmol/L, FT3:1.9 pmol/L).MediumⅢ, DMEM with 5 nmol/L T3+ 15% low TH FBS (TT3 4.3 nmol/L, FT3 23.2 pmol/L).1.2 Cell Culture and GroupingCerebral cortex was obtained from 1-2 d age healthy SD rat under sterile conditions. Then, washed with Hanks solution, shredded, trypsin digestion and cultured in medium. The astrocytes were identified with anti-GFAP antibody at 8th day, and they were divided into 4 groups according to the different medium.Control group:mediumⅠfor 2 days + mediumⅢfor 6 daysHypothyroidism group:mediumⅠfor 2 days + mediumⅡfor 6 daysIntervention group 1:mediumⅠfor 2 days + mediumⅡfor 2 days + mediumⅢfor 4 daysIntervention group 2:mediumⅠfor 2 days + mediumⅡfor 4 days + mediumⅢfor 2 days1.3 Parameter MeasurementsTetrazolium blue (MTT) method was used to draw the growth curve. The expression of Cx43, Cx32 and Cx45 mRNA in the cultured cells were detected by using the Takara Real time RT-PCR kit. The levels of Cx43, Cx32 and Cx45 protein were measured by Western blot, and laser scanning confocal microscope was used for locating the protein in the cells.2 ResultsThe cell proliferation was slow and exponential growth phase was right shift in the hypothyroidism group, and improved after the intervention. The expression of Cx43 in astrocyte decreased in the hypothyroidism group. After T3 intervention, the Cx43 levels had an upward trend. They mRNA levels of Cx43 can recovry to normal levels while the protein levels did not return to normal levels. Laser scanning confocal microscopy showed that abundant Cx43 protein expressed astrocytes, including the intracytoplasm of body and dendritic. There were many well-distributed high- fluorescence intensity points between adjacent astrocytes in control group while the point structures in hypothyroidism group were less with low fluorescence intensity. After T3 intervention, the point structures were increased, but asymmetrical distribution.The mRNA level Cx32 in the hypothyroidism group was lower than that in the controls, and it can be rapidly increased to near normal levels after T3 intervention. However, the protein was undetectable both using Western blot and laser scanning confocal microscopy.A low Cx45 level was noted in the hypothyroidism group as well, and it can return to normal levels with early intervention. Laser scanning confocal microscopy showed that abundant Cx43 protein expressed astrocytes, including the intracytoplasm of body and dendritic. There were only few high- fluorescence intensity points between adjacent astrocytes.Conclusions1. The levels of Cx43 decrease and the levels of Cx32 increase in the cerebral cortex of CH mice.2. Low TH induces low Cx43 and Cx45 expression, and the abnormality of GJ distribution in astrocytes.3. The regulation of TH on gene transcription has a spatial and temporal control.4. Short-term T3 early intervention increases the Cx43 and Cx45 expression, but the levels of Cx 43 does not recovery to normal levels.