Influence Of ProBDNF On Survival And Neurite Outgrowth From Rat Spiral Ganglion Neurons

Objective:The aim of this work was to establish a reproducible spiral ganglion neuron culturing system in vitro. Then counted the surviving number of spiral ganglion neurons and observed their neurite outgrowth in this system. We also compared the effects of different concentrations of precursor brain derived neurotrophic factor on the survival and neurite outgrowth of cultured neurons. To study the influence mechanism of proBDNF, we observed the expression of p75NTR in spiral ganglion neurons from different groups and evaluated the role of c-Jun N-terminal kinase signaling pathway in spiral ganglion neurons.Methods:Spiral ganglions tissues were collected from postnatal day 3-5 Sprague Dawley rats after sacrificed. By explants culture method, spiral ganglion explants were plated on eight-well chamber plates which were coated by laminin/poly-d-lysine and then incubated for 24 hours. By dissociated culture method, spiral ganglion explants were enzymatically digested and suspended. Then dissociated spiral ganglion cells were plated on coated eight-well chamber plates and maintained at 37℃ for 4 hours to promote the attachment of the neurons. Cultured dissociated spiral ganglion cells were randomly divided into five groups:control group, BDNF group(BDNF,10 ng/ml), C10 group(proBDNF,10 ng/ml), C50 group(proBDNF,50 ng/ml), C100 group(proBDNF,100 ng/ml).48 hours after incubation spiral ganglion neurons were fixed and stained forTUJl. Counted all neurons and observed their neurite outgrowth under fluorescence microscope. Statistical analyses for the number of surviving neurons by using oneway analysis of variance to compare each proBDNF subgroup with the untreated control group.Results:Cultured spiral ganglion explants were firmly adherent after 24h incubation. Many neurites extended from explants edge. The dissociated spiral ganglion neurons in explants culturing system were surviving and grew well, but they were unevenly distributed. However, the dissociated spiral ganglion neurons in dissociated culturing system were evenly distributed and grew well. I mmuno fluorescence staining showed that the surviving number of spiral ganglion neurons per well was 79.8±5.3 in the control group,82.3±6.0 in the BDNF group,69.7±6.3 in the C10 group,54.3±4.6 in the C50 group and 22.7±4.1 in the C100 group. Compared with the control group, C50 group and C100 group had statistically significant survival-reducing effects (P<0.001). Furthermore, surviving numbers of the three groups with proBDNF were lower than the BDNF group. In order to assess the effect of proBDNF on cell morphology, spiral ganglion neurons were divided into two categories:spiral ganglion neurons with or without neurites. The results demonstrated that proBDNF significantly enhanced the proportion of spiral ganglion neurons without neurites in C10, C50 and C100 groups compared with control group(P<0.001). In addition,20 μM SP600125 which is the c-Jun N-terminal kinase inhibitor was added to the C50 group, the surviving number of spiral ganglion neurons increased to 91.5±8.2/well.Conclusions:Two methods both cultured spiral ganglion neurons successly, but dissociated culture method is more suitable than explant culture method to study the influence of proBDNF. In cultured spiral ganglion neuron, proBDNF promote apoptosis of neurons and inhibits the sprouting of neurites by p75NTR-JNK signaling pathway, inhibition of JNK signaling attenuated the effect of proBDNF on spiral ganglion neurons’ survival.