Neuron‐Enriched cultures of adult rat dorsal root ganglia: Establishment, characterization, survival, and neuropeptide expression in response to trophic factors

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Abstract
It is unknown whether adult dorsal root ganglion (DRG) neurons require trophic factors for their survival and maintenance of neuropeptide phenotypes. We have established and characterized neuron‐enriched cultures of adult rat DRGs and investigated their responses to nerve growth factor (NGF), ciliary neuronotrophic factor (CNTF), pig brain extract (PBE, crude fraction of brain‐derived neuronotrophic factor, BDNF), and laminin (LN).DRGs were dissected from levels C1 through L6 and dissociated and freed from myelin fragments and most satellite (S‐100‐immunoreactive) cells by Centrifugation on Percoll and preplating. The enriched neurons, characterized by their morphology and immunoreactivity for neuron‐specific enolase, constityted a population representative of the in vivo situation with regard to expression of substance P (SP), somatostatin (SOM), and cholecystokinin‐8 (CCK) immunoreactivities. In the absence of trophic factors and using polyornithine (PORN) as a substratum, 60‐70% of the neurons present initially (0.5 days) had died after 7 days.LN as a substratum did not prevent a 30% loss of neurons up to day 4.5, but it subsequently maintanied DRG neurons at a plateau. This behavior might reflect a cotrophic effect of LN and factors provided by non‐neuronal cells, whose proliferation between 4.5 and 7 days could not be prevented by addition of mitotic inhibitors of γ‐irradiation.CNTF, but not NGF, slightly enhanced survival at 7 days on either PORN or LN. No neuronal losses were found in non‐enriched cultures or when enriched neurons were supplemented with PBE, indicating that non‐neuronal cells and PBE provide factor (S) essential for adult DRG neuron survival. Proportions of SP‐, SOM‐, and CCK‐immunoreactive cells were unaltered under any experimental condition, with the exception of a numerical decline in SP cells in 7‐days cultures with LN, but not PORN, as the substratum.Our data, considered in the context of recent in vivo and vitro studies suggest that a combination of tropic factors or an unidentified factor, rather than the established NGF, CNTFl, and BDNF, which address embryonic and neonatal DRG neurons, are required for the in vitro maintenance of adult DRG neurons.

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