The Effect And Underlying Mechanism Of Olfactory Ensheathing Cells On Spiral Ganglion Cells In Vitro

Part 1 Culture, purification and identification of olfactory ensheathing cellsObjective:To culture and purify olfactory ensheathing cells(OECs). Methods:The olfactory nerve layer of adult rat olfactory bulb was dissected. The tissues were minced with a razor blade, and trypsinized using trypsin. OECs were obtained and purified based on their special rate of attachment which was different from the other harvested cell types during culture. DF12 medium supplemented with 10% fetal bovine serum and antibiotics was employed to culture the OECs. Basic fibroblast growth factor (bFGF,20ng/ml) and forskolin (2uM) were added to the medium to enhance OECs growth two days after plating. Half of the medium in each group was refreshed twice a week. OECs were immunocytochemically characterized and confirmed by expression of low-affinity nerve growth factor receptor P75NTR and glial fibrillary acidic protein (GFAP). Results:OECs from the olfactory nerve layer of adult SD rats were highly purified based on the differing rates of attachment of the various cell types. Cultured OECs displayed a typical spindle-shaped soma with long and slim processes. The addition of bFGF and forskolin two days after plating can activate the growth of olfactory ensheathing cells and single layer of OECs was seen seven days after plating.92 percent of the cells cultured were P75NTR immunoreactive, and about 12 percent of the cells positive for GFAP. Conclusion:The high pure of olfactory ensheathing cells could be harvested using special rate of attachment which was different from the other harvested cell types during culture and facilitate the research on the olfactory ensheathing cells in vitro. Addition of basic fibroblast growth factor and forskolin into the medium two days after plating could promote the growth of olfactory ensheathing cells. Objective: To explore whether olfactory ensheathing cells (OECs) can promote newborn rat spiral ganglion cells (SGCs) survival. Methods:Co-cultures of OECs from adult rat olfactory bulb with SGCs from newborn rat cochlea were established and single culture of SGCs acted as control. In addition, OECs conditioned medium (OEC-CM) was employed to culture SGCs in contrast with the co-cultures. OECs were obtained and purified based on their special rate of attachment which was different from the other harvested cell types during culture. OECs and SGCs were immunocytochemically characterized and confirmed by expression of low-affinity nerve growth factor receptor P75NTR and neuron-specificβⅢ-tubulin, respectively. For statistic analyses, randomized block ANOVA was performed. Results: Single layer of OECs (92% pure) was seen seven days after plating. Surviving SGCs, that extended their primary neurites, were found on the surface of the layer in the co-cultures. When OECs and SGCs were co-cultured, the number of surviving SGCs was significantly greater than that in other two groups (P<0.01). Nine days after culture, there was even no change in the number of surviving SGCs in co-cultures while the number of SGCs in single culture dramatically reduced. The number of surviving SGCs in control group significantly decreased from 14.6±1.1 SGCs/ field on day 3 to 3.3±0.3 SGCs/ field on day 14. By contrast, OEC-CM had a positive effect on SGCs survival, with an overall decrease of surviving SGCs from 35.3±2.1 SGCs/field on day 3 to 12.9±0.3 SGCs/ field on day 14 of culture. Surprisingly, the number of surviving SGCs co-cultured with OECs slightly decreased from 66.9±1.2 SGCs/ field on day 3 to 38.9±1.0 SGCs/ field on day 14. Conclusion:Olfactory ensheathing cells can promote the survival of newborn rat spiral ganglion cell in vitro. Objective:To explore the underlying mechanisms that OECs promote the survival of SGCs in vitro. Methods:Co-cultures of OECs from adult rat olfactory bulb with SGCs from newborn rat cochlea were established. In the present study, there were three groups:co-cultures, co-cultures with addition of BDNF (500 pg/ml), and co-cultures with addition of anti-BDNF antibody (50μg/ml). The cultures were fixed three days after cultured. SGCs were immunocytochemically characterized and confirmed by the expression of neuron-specificβⅢ-tubulin. One-way ANOVA and Bonferroni were employed to investigate the differences in the number of SGCs in different cultures. Results:In comparison with co-cultures without treatment, addition of BDNF (500 pg/mL) into the medium had no obvious survival-promoting on SGCs. The number of surviving SGCs reduced significantly when anti-BDNF antibody(50μg/mL) was applied into co-cultures (P<0.01). The number of SGCs between co-cultures and co-cultures+BDNF has no significant difference (P>0.05); the difference between co-cultures+anti-BDNF and the others was significant (P<0.01). Conclusion:BDNF expressed by OECs is in part responsible for the survival-promoting of olfactory ensheathing cells on spiral ganglion cells.