Apoptosis in Primary Cultures of E14 Rat Ventral Mesencephala: Time Course of Dopaminergic Cell Death and Implications for Neural Transplantation

Abstract

Transplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum. However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery. A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear. Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival. Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media. After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically. Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h. Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons. There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h. We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.