| Part I The culture and identification of spinal motoneurons in vitroObjective: To explore the dissociation, the purity and the culturing method in vitro of spinal motoneurons and to find out an effective way to identify this kind of cells. Spinal motoneurons culturing in vitro serve as an available tool to further study ALS especially the degeneration and death about motoneurons.Methods: Spinal motoneurons were isolated from spinal cords of El6 rat embryos by several steps including digestion, density gradient centrifugation on 6.8% metrizamide cushion and so on. Purified motoneurons were obtained by collecting the layer on the top of metrizamide cushion and letting glial adhere first. In the end, the enriched motoneurons were plated in 24-well plates at a density of 4X 105/ml. Arabinosylcytosine were added to culturing system after 24 hours in order to inhibit outgrowth of glial. L-15 serum medium was used for the first 48 hours and then was changed with serum-free medium. Later, neurons were refeeded with 50% exchange of medium every two days. Spinal motoneurons were identified by being marked with polyclonal antibody choline acetyltransferase (ChAT) according to ABC method.Results: Spinal motoneurons were the cells which were immunoreactive to antibodies against ChAT. My outcome revealed that ChAT-positive cells were more than 85% of all culturing cells. The life time of spinal motoneurons was from seven to nine days becausethey couldn't proliferate. Serum-medium and serum-free medium were combined to increase the purity of spinal motoneurons and improve their survival time.Conclusions: This study provided one method to culture spinal motoneurons successfully. Spinal motoneurons in vitro could be an ideal model to study some injuring or protective mechanisms of motoneurons. Spinal motoneurons could be identified by being marked with polyclonal antibody ChAT.Part II Morphometric analysis of spinal motoneuronsObjective: We grasped the surival rate and the morphometric properities of motoneurons at different culturing time, so we understood their growing regulations .We knew what's time to add experimental factors and what's time to get outcome according to these findings.Methods: the number of spinal motoneurons was counted at different culturing time (such as 0; 1; 3; 5; 7; 9 days) under microscope at 250 X. Cell number at the beginning of plating was made as a base line 100%. The surival rate of every group was shown by percentage. ChAT-positive neurons were chosen as being analysized subjects. Five indexes such as axonal length, total dendrites length, number of bifurcation, dendrites number and cell body area were measured using image analysis system.Results: Survival rate of spinal motoneurons trended to decrease with time passing by. It dropped to 59% for the first day and maintained at the rate of 50% following three to five days. The survival dropped gradually to 37% at the seventh day. Most of neurons had been dead after nine days. When it concerned to morphology, not only axonal length , dendrites length, dendrites number but also cell body area increased rapidly for the first three days, and improved steadily from the fourth day to the seventh day , then decreased sharply after culturing seven days. All neurons were going to die with body withering and dendrites rupturing at the ninth day.Conclusions: We could observe that the growing condition of spinal motoneurons through these following indexes: survival rate, axonal length, dendrites length, dendrites number, the number of bifurcation and cell body area. Neurons growing from the third day to the fifth day could be chosen to measure their morphometric indexes because motoneurons had grown best in this period.Part III Effects of GSH and L-NAME on spinal motoneurons in vitroObjective: This experiment was undertaken to explore whether oxidative damage attributed to the degeneration or death of motoneurons and to study whether GSH or L-NAME had protective effects on motoneurons in vitro.Methods: Th... |
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