Study On The Characterization Of Development In Neonatal Bovine Retinal Neurons In Vitro

PWith the development of retinal transplantation, the selection of donor for retinal transplantation has been paid more and more attention. However, it is difficult to acquire a large number of human retina for transplantation. Now people try to store dissociated fetal and neonatal retina in vitro and set up donor bank for retinal transplantation. Because of the developmental characterization of fetal and neonatal retinal neural cells, the age of the donor and the storage time in vitro has been the key factor for the selection of donor. In this study, a culture system has been found for neonatal bovine retinal neural cells. The characterization of morphology and immunocytochemistry at different culture stage had been studied. The electrophysiological characterization of the cultured rod had been studied by whole cell patch clamp. The best culture stage of retinal neurons has been decided. The result of this study will provide theoretical basis for the selection of retinal transplantation donor.1. Study on the development of morphological characterization in retinal neuronsNearly all cells showed a circular outline and lacked cell process just after attachment. With the increase of the culture time, some cells began to extending processes. The neural cells showed different morphological characterization. The neurons cultured for 20 days in vitro had mature morphology. There were three kinds of neurons according to the morphological character, process-free cells, multipolar neurons and cells with apical process. There were broad diversity in the size of the cell body and number, extension and branching patterns of nerve fibers from different neurons, which was the symbol of development for neurons in vitro. Different neurons contacted with each other by neurite. Growth cones were always seen at the end of the neurite, which indicated that the cultured neurons were in good developmental state. After 30 days in vitro, multipolar neurons trended to degenerate in the way of decrease luster of the cell body and decrease in numbers. Cells with apical process and process-free cells were not affected until 40 days in vitro. At 90 days in vitro, the cell body of neurons were crimpy and the processes shrank. The cells soon fall off the base and died.Low concentration of fetal bovine serum in culture medium had been choose for this experiment. It can provide necessary growth factors for growth and proliferation of the cells, at the same time, the inhibition substance in the serum had been decreased. Low concentration of fetal bovine serum can inhibit the excess proliferation of the glial cells, which is good for the growth of neurons. Mixed culture system had been selected for this experiment(Ofand glial cells had been maintained in the culture, dial cells can secrete necessary cellular factors for neurons. Different neurons can contact with each other and pass information in this culture system. This culture system is less complicated than purified culture. It did not need to add growth factors in the culture and neurons can survive for a long time in vitro. 2. Study on the development of immunocytochemical characterization in different neuronsSpecific antibodies had been used as marker for different neurons, Calbindin-D28K for retinal horizontal cells, PKC for rod-bipolar cells, CD90 for retinal ganglion cells(RGCs), Rho4D2 for rods, GFAP for glial cells. The calbindin-D28K positive neurons showed immature morphological character at 10 DIC (days in culture). Neurons cultured for 20 days in vitro presented mature morphological character of long and more number of neurite, which was same as the mature horizontal cells. The immunoreactive intensity of positive neurons at 20 DIC was highest compared with that at other culture stage. The immunoreactive intensity of stained neurons deceased markedly at 30 DIC and 40 DIC, indicating that the positive cells turned to be senile. The retinal horizontal cells cultured for 20 days showed mature morphological and immunoreactive characterization. The best culture time for...