Spontaneous association of glial cells with regrowing neurites in mixed cultures of dissociated spiral ganglia

Abstract

Evidence from developmental and regeneration studies of the cochlea and other tissues gives reason to hypothesize a role for nonneural cells in the growth and regeneration of cochlear spiral ganglion nerve fibers. We examined the spontaneous associations of regrowing neurites and nonneural cells in mixed cultures of dissociated newborn mouse spiral ganglia. After 7 days in vitro, nonneural cells formed a confluent layer in the culture well. Regrowing neurites grew atop this layer, forming non-uniform patterns that were similar to those formed by endogenously expressed laminin-1, entactin and integrin β4, but not fibronectin or tenascin. In cultures grown for 42 h and maintained in three different growth media, all regrowing neurites were preferentially associated with spindle-shaped nonneural cells. The spindle-shaped cells incorporated bromodeoxyuridine in culture and were immunoreactive for the proteins S100, laminin-1, laminin-2, SRY-related high-mobility-group box 10 transcription factor (Sox10), neurotrophin receptor (P75) and connexin29 but negative for fibronectin and glial fibrillary acidic protein. These cells existed in the culture within a much larger, general population of fibronectin positive cells. Immunolabeling of fixed cochleas from neonatal mice localized Sox10, P75 and connexin29, to peripheral nerve bundles. The observed expressions of protein markers and the bipolar, spindle shape of the neurite-associated cells indicate that they are derived in vitro from the original Schwann or satellite cells in the ganglion or spiral lamina. The spontaneous and preferential association of neurites in culture with mitotic Schwann cells highlights the potential contribution neurite–Schwann cell interactions may have in promoting the growth and regrowth of damaged spiral ganglion neurons in the cochlea.