gms | German Medical Science

Auditory neuropathy is a sensorineural hearing disorder wherein the outer hair cells are functional but the transmission of the acoustic information to the brainstem is lacking. Since spiral ganglion neurons are crucial for sending the generated nerve impulses from the hair cells to the brainstem, a reliable method to culture them in vitro might open new possibilities to study auditory neuropathy. Cultured neuronal cells are highly dependent on neurotrophic factors, which are secreted by non-neuronal cells like glial cells. Since the secretion of growth factors by these cells is not controllable in vitro, the aim of this study was to develop a glial cell free culture for spiral ganglion neurons.

Cells were isolated from the spiral ganglion of C57Bl/6 mice at postnatal day 5 and cultured on coverslips with different coatings. Various cell culture media and factors were tested. After 4 days cultured cells were fixed and stained by DAPI, β-Tubulin and β-III Tubulin. Number of glial cells and neurons were evaluated as well as neurite length.

No significant difference in glial and neuronal cell number between Polyornithin and Poly-D Lysine as coating material could be evaluated. However, additional laminin coating increased significantly axon length and raised the ratio between neurons and glial cells. Furthermore the use of DMEM medium increased the percentage of neurons compared to Neurobasal medium. Arabinosylcytosine as additive decreased the glial cell number, but increased the numbers of neurons whereas neurite length was not affected. Addition of leukemia inhibitory factor, a pleiotropic cytokine which plays a role in survival of sensory and motoneurons, increased the percentage of neurons in culture, too.

In conclusion Laminin coating, the use of DMEM Medium and addition of Arabinosylcytosine and leukemia inhibitory factor results in nearly glial cell free culture conditions for in vitro experiments of spiral ganglion neurons. These optimized conditions will prove to be useful to further investigate the molecular properties of the cultured auditory neurons without uncontrollable endogenous stimulation. This could possibly lead to new insights regarding the pathophysiological findings in auditory neuropathy.