Studies of cell death in Parkinson’s disease using organotypic cell cultures.

Abstract

In this study we aimed to investigate the effects of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and rotenone neurotoxins on dopaminergic (DAergic) neuronal survival using ventral mesencephalic (VM) organotypic cell culture derived from postnatal rat pups (P4-5) and immunocytochemistry for tyrosine hydroxylase (TH) as a marker of DAergic cells. In addition, we examined the neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) on TH-ir cells exposed to MPTP and rotenone as a possible treatment for PD. The TH-ir cells in co-cultures with striatum (ST) as a target grew better then when VM was cultured alone and that TH-ir cells in co-cultures could be maintained without using conditioned and trophic media. We treated 7 day and 14 day co-cultures at different times with varying MPTP and rotenone concentrations and found 14 day old cultures were more vulnerable than 7 day old co-cultures to the effects of either neurotoxin with TH-ir cell numbers significantly lower in 14 day cultures compared to 7 day cultures. Both neurotoxins induced a dose-dependent TH-ir cell reduction in the co-cultures. In addition we compared the toxicity of MPTP and its active metabolite 1-methyl-4- phenylpyridinium (MPP+) as the neurotoxic effects of MPTP on DAergic cells depends on its conversion to MPP+ by astrocytes. We found no significant difference in TH-ir cell reduction in co-cultures treated with MPTP and MPP+. Rotenone was more toxic than MPTP with less TH-ir cell survival in the weeks post treatment. GDNF exposure produced increased cell size and significant increases in TH-ir cell branching in cocultures in a dose-dependent manner. Post treatment of GDNF against MPTP and rotenone provided significant neuroprotection as TH-ir cell survival was at the lower neurotoxin doses and not at the higher doses.