Effects of antioxidant pretreatment on the survival of embryonic dopaminergic neurons in vitro and following grafting in an animal model of Parkinson's disease.

The effect of pretreating cell suspensions of embryonic rat ventral mesencephala (VM) with antioxidant combinations on the survival of dopaminergic (DA) neurons was studied in vitro and following transplantation into the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease. The in vitro experiments examined the effects of two thiol antioxidants, N-acetyl-L-cysteine (NAC) and reduced glutathione (GSH), and a member of the lazaroid family of 21-aminosteroids, U-83836E, singly and in combination, on survival of DA neurons derived from dissociated E14 rat VM tissue. For in vivo studies, cell suspensions were pretreated with combinations of NAC, GSH, and U-83836E prior to transplanting into 6-OHDA-lesioned rats to investigate whether DA neuron survival could be further improved. NAC, GSH, and U-83836E individually increased DA neuron survival in vitro and a combination of all three resulted in the greatest survival. In vivo, pretreatment with U-83836E alone resulted in a significantly greater reduction in amphetamine-induced rotation 6 weeks postgrafting compared with a control group receiving nontreated graft tissue. This functional effect correlated with a significant improvement in DA neuron survival 6 weeks postgrafting. The thiol combination pretreatment of NAC and GSH, and the triple combination of NAC, GSH, and U-83836E, however, failed to improve both functional recovery and DA neuron survival when compared with the nontreated control grafts.

Neuronal nitric oxide synthase immunohistochemistry and 4,5-diaminofluorescein diacetate: tools for nitric oxide research.

Nitric oxide plays an important role in many biological processes including the cardiovascular, immune, reproductive and nervous systems. There is much interest surrounding this molecule and the enzyme responsible for its synthesis, nitric oxide synthase. In order to investigate the role of nitric oxide in various biological processes it is necessary to be able to identify the released molecule itself as well as nitric oxide synthase. Detection of nitric oxide synthase was achieved by optimisation of the immunohistochemical localisation of the neuronal isoform of the enzyme in primary cultures of rat ventral mesencephala and in fixed adult rat brain sections. Furthermore, using 4,5-diaminofluorescein diacetate, we have directly visualised endogenously produced nitric oxide in mesencephalic cultures and demonstrated the potential use of this indicator for visualising nitric oxide produced in vivo.

Apoptosis in primary cultures of E14 rat ventral mesencephala: time course of dopaminergic cell death and implications for neural transplantation.

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.

cAMP included during cell suspension preparation improves survival of dopaminergic neurons in vitro.

The physical process of cell suspension preparation from embryonic ventral mesencephala (VM) may be responsible for the low numbers of dopaminergic (DA) neurons that survive following neural transplantation or in vitro culture. In particular, the disruption of cell to extracellular matrix attachment may result in cell death through deactivation of a cAMP-dependent protein kinase involved in cell survival signalling. In an attempt to reduce this death, dibutyryl cAMP was included in all solutions from explant collection to final dissociation. Pretreatment with 700 microM dibutyryl cAMP resulted in 90% survival of the DA neurons originally plated, compared with only 40% in the untreated cultures, after 5 days in vitro. Treatment of VM explants in this manner may result in major improvements in neural transplantation as a technique for the treatment of Parkinson's disease.