A Developmental Study On The NTs And Receptors Expressions In The Human Embryo Spinal Cord

Objective To explore the relations between the neurotrophins family and itsreceptors and the human embryonic spinal cord development.Method The expressions and changes of neurotrophins family and its receptorswere studied systematically in human embryonic spinal cord from the 3 ^rd week to 8^th month by the methods of Immunohistochemistry , In Situ Hybridization, Western Blotting and Reverse Transcription Polymerase Chain Reaction (RT-PCR).Result 1. From 3^rd week to 4^th week, NGF protein was expressed at medullaryepithelium, and it existed in the prosoma of nerve cells of mantle layer, basal plate and alar plate from 5^th week to 7^th week, and positive immunoreaction increased along with embryonic development. From 8^th to 9^th week, massive positive cells, whose density in alar plate was higher than that of basal plate, were seen at mantle layer. Meanwhile, all kinds of neuroblasts appeared gradually. Although the density of positive cells decreased after 3^rd month, mature nerve cells were seen obviously and its number increased gradually in the gray matter. After 6^th month the shape of spinal cord and positive nerve cells in the gray matter tended to be mature. Positive nerve fibers were seen at cortex-medulla border at ^th week. The gliocytes appeared in white matter at 3^rd month and increased at 6^th month. Usually, gliocyte's differentiation was later than nerve cell's, and positive nerve fiber appearance earlier than the gliocyte in the white matter. The results of Western Blotting demonstrated that NGF protein content increased gradually along with the embryonic age from 6^th to 8^th week, reaching the peak at 8^th or 9^th week(P<0.05 )and beginning to decrease from 3^th month (P<0.05) ,after that it increased again after 6^th month, and then maintained relatively steady. The result of protein content was the same as the protein localization changes of immunohistochemistry. The result of in situ hybridization showed that NGF mRNA took on weak positive expression at 4^th week at medullary epithelium and was later than that of protein. Positive expression increased afterwards along with the development of spinal cord until it was seen in all kinds of neuroblasts at 8^th week or 9^th week and dropped at 3^th month. Positive multipolar neurons were can't be seen until 4^th month. The shape of positive nerve cells tended to be mature from 6^th month to 8^th month. The positive immunoreaction of nerve fiber and gliocyte is later to the nerve cell's in the white matter. Its expression pattern was similar to the NGF protein'. The result of RT-PCR showed that NGF mRNA was gradually upregulated from 6^th to 8^th week, reaching the peak at 8^th week (P<0.05) . Its expression was downregulated after 9^th week, declining to the minimum value in 4^th month (P<0.05) . Later it will be upregulated gradually again. The NGF mRNA content measurement result was identical to localization expression changes of in situ hybridization. 2. TrkA protein was seen at medullary epithelium and internal limiting membrane at the end of 3^rd week and 4^th week. It was expressed in kytoplasm at mantle layer, basal plate and alar plate from 5^th week to 7^th week along with the medullary tube growth, and from 8^th to 9^th week positive cells with karyolemma and nucleoli concentrated, whose density in alar plate was higher than that of basal plate. Gradually, all kinds of differentiating positive neuroblasts appeared. From 10^th week to 3^th month, the number of big multipolar neurons increased gradually, so did gliocyte in white matter. There were many positive neurons in dorsal horn and ventral horn, and positive gliocytes and fibres in the white matter, which tended to be mature after 6^th month. The result of Western blotting demonstrated that TrkA protein had certain content at 6^th week, but wasn't much. Its content increased slowly from 6^th week to 3^th month, but increased obviously in 4^th month. The content of TrkA protein had dropped in 5^th month, but increased slightly in 7^th month. The change tendency was identical to that of immunohistochemistry localization expression. The result of in situ hybridization showed that TrkA mRNA took on weak positive expression at 4^th week at medullary epithelium, and was later than that of protein. Positive expression increased afterwards along with the spinal cord growth until it was seen in mantle layer, basal plate and alar plate. At the same time, positive immature apolar neuroblasts were turned into all types of neurons. A few multipolar neuroblasts were seen until 9^th week and its number enlarged gradually. The expression mode was similar to the TrkA protein'. The RT-PCR result showed that TrkA's mRNA expression was tested in 9^th week, reaching the peak in 3^th month (P<0.05 ) . Then its expression was downregulated , but the fluctuation wasn't big. 3. BDNF protein was seen at external and internal limiting membrane of medullary epithelium at the end of 3^thweek and 4^th week and had a stronger positive immunoreaction. BDNF protein expression position was similar to NGF protein's. But it was expressed obviously in prominence of neuroepithelial cells in earlier time, and trachychromatic fasciculus was seen in the white matter besides all kinds of nerve cell in the gray matter. The result of Western blotting demonstrated that the BDNF protein content was indentical to the change of immunohistochemistry localization expression, whose change curve was similar to NGF's. The result of in situ hybridization showed that BDNF mRNA was expressed obviously at medullary epithelium at the end of 3^th week and 4^th week. Its expression mode was similar to that of protein, and the positive reaction was stronger than NGF's. The BDNF mRNA content measurement was identical to localization expression change of in situ hybridization. 4. The result of immunohistochemistry showed that TrkB protein expression took on weak positive at external and internal limiting membrane of medullary epithelium and mantle layer at 5^th week. The expression position was similar to the BDNF protein' in the after development time of spinal cord, but its positive reaction was obviously weaker than BDNF protein's. The protein quantitation demonstrated that TrkB's expression tendency was in an upregulation state from 6^th week to 4^th month, but no obvious upregulation time. Its expression dropped after 4^th month and upregulated slightly since 6^th month but the level was very low in the whole. The TrkB protein expression was identical to the expression change of immunohistochemistry. The result of in situ hybridization showed that TrkB mRNA was expressed at medullary tube at 5^th week, whose expression mode was similar to that of TrkB protein expression. The RT-PCR result showed that TrkB mRNA was upregulated gradually along with the development of spinal cord in the early time, reaching the peak at 8^th week and decreasing from 3^th month, which was similar to BDNF's expression change. 5. NT-3 protein expression was characteristic, positive reaction was mainly concentrated on cell process and fiber. NT-3 protein expression took on strong positive at medullary epithelium at 3^rd or 4^th week, and the positive cell process own a radial shape. There were massive radial fibers in the neuroepithelial layer and mantle layer from 5^th to 8^th week, and prosoma of nerve cell from 8^th to 11^th week was much too. The positive reaction position was similar to NGF and BDNF's. Western Blotting analysis showed that NT-3 protein reached a certain level from 6^th week, rose gradually to the peak at 9th week (P<0.05) , and then decreased and maintained a certain level, which was identical to localization expression change. The result of in situ hybridization showed that NT-3 mRNA took on weak positive expression at medullary epithelium at 4^th week, which is later than protein expression. Its expression mode was similar to that of protein expression, but it was weaker than that of protein in cell process. The RT-PCR result showed that the NT-3 mRNA expression change was identical to the localization expression change. 6. The immunohistochemistry result showed that TrkC protein expression took on weak positive at internal limiting membrane of medullary epithelium at 3^rd week, whose expression mode was similar to that of NT-3. But it was weaker than that of NT-3 in the cell process and stronger than that of TrkA and TrkB. The Western Blotting result matched that of localization expression change. TrkC mRNA was expressed in partial cells of neurepithelium at 4^th week. Its expression position was similar to that of TrkC protein at 5^th week, but it is weaker. The RT-PCR result showed that TrkC mRNA content change was indentical to localization expression change. 7. NT-4 protein took on the weak positive expression at medullary epithelium at 3^rd week, whose expression mode was similar to that of NGF and BDNF. There were more positive neurons in the anterior angle than that of intermediate zone and dorsalhorn. Positive gliocytes were more in posterior cord and lateral cord than that of anterior column in the white matter. Result of Western Blotting showed that NT-4 had existed at 6^th week and increased slowly afterwards , reaching the peak at 9^th week (P<0.05) . Later, it dropped slowly and maintained steady at a certain level in 5^th and 6^th month. NT-4 protein content change was basically consistent with morphological change of immunohistochemistry. NT-4 mRNA was located in medullary epithelium at 4^th week, which was similar to the expression of its protein. The RT-PCR experiment result showed that the NT-4 mRNA content change was basically consistent with morphological change of in situ hybridization. 8. In the examined time intervals, all of the factors were expressed partly in ventricular cell and ependymal epithelium cell in different cell cycle of medullary epithelium.Conclusion 1. The Neurotrophins family's numbers, including NGF, BDNF,NT-3 and NT-4, was extensively distributed in all kinds of structures in different periods of embryonic spinal cord development and their expressions were stronger in spinal cord in the early time, which were overlapping sometimes and different sometimes. These evidences indicated that Neurotrophins family played a vital role in the human embryonic spinal cord development especially in the embryonic stage. However, they played different roles respectively in the different regions and cells. 2. The mRNA of Neurotrophins family's numbers existed widely in nerve cells and gliocytes in each development stage, which indicated that nerve cells and gliocytes of medullary tube or spinal cord had the capacity of synthesizing nerve nutrient by themselves. 3. Neurotrophins family's high-affinity receptors were widely distributed in nerve cells and gliocytes in each development stage, which indicated that Neurotrophins family members fulfilled their each physiological function by the method of autocrine and paracrine. 4. The Neurotrophins family possibly had the ability of inducing ependymal epithelium neural stem cells to carry on division growth.