Untersuchungen zu trophischen und protektiven Effekten neurotropher Faktoren (Brain-derived neurotrophic factor, Glial cell line-derived neurotrophic factor), des Glukocorticoids Dexamethason sowie elektrischer Stimulation auf kultivierte Spiralganglienzellen der Ratte

An optimized cochlear implant prosthesis therapy of deaf patients is dependend on a preferably great number of vital spiral ganglion cells. In this context various studies have been demonstrated that neurotrophic factors like GDNF and BDNF as well as electrical stimulation are promising therapeutic options to improve the survival of spiral ganglion cells and partly to induce an enhanced outgrowth of spiral ganglion neurites. With regard to their anti-proliferative and anti-inflammatoric effects glucocorticoids like dexamethasone are potentially suitable to reduce unintended side-effects of electrode implantation such as growth of connective tissue around the electrode. Terefore glucocorticoids are promising agents to improve the nerve-electrode contact when locally applied into the inner ear. So far it is unknown which concentrations and combinations of the mentioned factors are optimal with respect to an improved spiral ganglion cell survival and neurite outgrowth in vivo and in vitro. Therefore, the main emphasis of this study was put on following questions: 1. Spiral ganglion cells were microscopically dissected from 3-5 day old rats, mechanically/ encymatically dissociated and cultivated for 48 h. By application of the neurotrophic factors BDNF, GDNF and the glucocorticoid dexamethasone in different concentrations (25, 50, 100 ng/ml) and combinations (100 ng/ml) possible toxic, protective and trophic effects were investigated. After the cultivation period cells were fixated and immunocytochemically stained (avidin biotin complex method; primary antibody: anti-neurofilament 200 kD) in order to count vital spiral ganglion cells and measure outgrowing neurites afterwards. A BDNF concentration of 50 ng/ml was demonstrated to be maximum effective for an enhanced spiral ganglion cell survival as well as neurite outgrowth in vitro. In case of GDNF a concentration of 100 ng/ml could be identified to be effective for an increased spiral ganglion cell survival, whereas a combination of BDNF and GDNF (100 ng/ml) exerts no additional protective or trophic effect on spiral ganglion cells. Dexamethasone had no influence on spiral ganglion cell survival or neurite outgrowth in all concentrations investigated. A combination of dexamethasone with BDNF and GDNF neither reduced nor enhanced the protective and trophic effects of the neurotrophic factors on spiral ganglion cells. 2. The question of a hypothetically altered gene and proteine expression of the neurotrophic factors BDNF and GDNF and their receptors in cultivated spiral ganglion cells with addition of the mentionend neurotrophic factors (100 ng/ml) was investigated using semiquantitative RT-PCR and indirect immunofluorescence. A proteine expression of the receptors GFRα-1, Ret und TrkB could be detected in the spiral ganglion cell soma after a cultivation period of 4 and 48 h in all investigated conditions. Additionally the TrkB receptor could be detected on outgrowing neurites after 48 h in vitro in the BDNF stimulated condition. Following RT-PCR gene expression of BDNF, GDNF and their correlating receptors could be detected in spiral ganglion cells in all investigated culture conditions. A relative comparison of the gene expression in spiral ganglion cells was demonstrated to be difficult to accomplish because of a variing number of spiral ganglion cells in the different culture conditions. 3. Using a newly developed and evaluated (pH, temperature, cytotoxicity on electrially stimulated fibroblasts) cell culture device for the electrical stimulation of spiral ganglion cells suitable stimulation parameters for a maximum spiral ganglion cell survival and neurite outgrowth were tested as single stimulation and in combination with the application of the neurotrophic factor BDNF (100 ng/ml). As a result of inital experiments (regarding pH and temperature behaviour in the culture medium under electrical stimulation) and flowcytometric investigation of electrically stimulated and propidiumiodide stained NHDF-fibroblasts, an appliable maximum voltage of 40 V was identified to be suitable for the cultivation of spiral ganglion cells in electrical fields. A single electrical stimulation at voltages of 6 and 30 V was demonstrated to be not effective for an enhanced spiral ganglion cell survival or neurite outgrowth under the applied stimulation parameters (48 h, biphasic rectangular pulses, 50 Hz, 1 s active, 19 s passive) when compared to an unstimulated control group. In contrast survival rates of combined electrically and BDNF stimulated spiral ganglion cells could be demonstrated to be significantly increased in comparison to the control group and tendencially but not significantly enhanced when compared with the survival rates attained by single BDNF application. Considering the demonstrated facts it could be concluded that dexamethasone exerts no toxic effects on cultivated spiral ganglion cells. By reason of its anti-proliferative and anti-inflammatoric effects dexamethasone is an interesting agent which should be looked at further in vivo in context of an optimized cochlear implant supply. For the neurotrophic factors BDNF and GDNF optimal in vitro concentrations could be identified and protective and in case of BDNF trophic effects on spiral ganglion cells could be approved. A combined application of both factors caused no additional advantage when compared with single BDNF and GDNF application. The developed cell culture device for an electrical stimulation of cultivated spiral ganglion cells provides the possibility to test and identify interesting parameters of a patterned electrical stimulation before in vivo experiments were carried out.