Adult Guinea Pigs Geniculate Ganglion Cells In Vitro And Spiral Ganglion Cells

Objective:To investigate anatomy of geniculate ganglion in adult guinea pig. To determine optimal in-vitro culture systems for guinea pig’s geniculate ganglion cells (GGCs) and spiral ganglion cells (SGCs) by characterizing the in-vitro growth of GGCs and SGCs under different culture systems.Method:Anatomise full path of facial nerve coursing through the temporal bone and dissect spiral ganglion of adult guinea pig under anatomic microscope. Digest the facial nerve and spiral ganglion tissue with trypsin or collagenase type Ⅰ. After enzymatic treatment, the remaining tissue is cultured following two procedures. One group is planted with a basic culture medium supplemented with high concentration of serum. Substitute the planting medium with a serum-free medium supplemented with a substitute of serum as soon as that most cells have adhered to the walls of culture plate is observed. The other group is incubated in a basic culture medium supplemented with low concentration of serum and mitotic inhibitor during the whole period of in-vitro culturing. Check the survival and appearance of GGCs and SGCs at day4,7,11and16following planting. Neurons are identified by immunofluorescence method.Result:Geniculate ganglion of adult guinea pig has no obvious enlargement in diameter compared with facial nerve trunk. Geniculate ganglion tissue doesn’t response to trypsin while spiral ganglion tissue is dispersed into single cells by this enzyme. Much more single cells are observed in the visual field after enzymatic treatment by collagenase type Ⅰ. In-vitro GGCs are little spindle cells with few bundle fine neurites. GGCs distribute in chainlike pattern. At day11disintegration of majority of GGCs is observed. In-vitro SGCs are big cells with large round nucleus. SGCs are irregular in shape having several neurites projecting from the body of cell. SGCs tend to gather around and the interconnections between neurons are apparent. At day11, degeneration of SGCs is more significant in the medium containing serum and mitotic inhibitor. The appearance of non-neuron cells is more profound in medium supplemented with serum.Conclusion:Geniculate ganglion of adult guinea pig is difficult to locate. GGCs and SGCs of adult guinea pig could survive in appropriate in-vitro culture condition. Tissue of geniculate ganglion and spiral ganglion could be digested with collagenase type Ⅰ while trypsin can only disperse the tissue of spiral ganglion. Both the medium with low concentration of serum and mitotic inhibitor and the serum-free medium supplemented with a substitute of serum are able to maintain the growth of GGCs and SGCs in vitro. Exist of low concentration of serum facilitates the growth of non-neuron cells. Mitotic inhibitor is able to prevent the multiplication of non-neuron cells to a certain degree but it also produce cytotoxic effect to neurons. Using serum-free medium not only can inhibit multiplication of non-neuron cells but also avoid the potential cytotoxic effect produced by mitotic inhibitor.