Effects of Fetal Mesencephalic Cell Grafts on the Intrastriatal 6-hydroxydoapmine Lesioned Rats

The effects of fetal mesencephalic cell grafts on the restoration of nigrostriatal dopaminergic function were studied in the intrastriatal 6-hydroxydopamine-lesioned rats. Four weeks after lesioning, transplantation of ventral mesencephalic cells from embryonic day 14 fetuses showed the number of tyrosine hydroxylase (TH) positive cells and fiber outgrowth in the grafted striatum, and significantly ameliorated symptomatic motor behavior of the animals, as determined by apomorphine-induced rotation. Furthermore, in substantia nigra pars compacta (SNc), the numbers of TH cells and fibers were markedly restored. Dopamine content of ipsilateral SNc was close to that of contralateral SNc (91.9 9.8%) in the transplanted animals, while the ratio was approximately 32% in sham-grafted animals. These results indicate that grafted cells restored the activity for the dopaminergic neurons located in SNc, although they were transplanted into striatum. In addition, we showed that the implanted fetal cells expressed high level of glial cell line-derived neurotrophic factor (GDNF), suggesting that the transplanted fetal cells might serve as a dopamine producer and a reservoir of neurotrophic factors. These results may be helpful in consideration of the therapeutic transplantation at early stage of PD.

Involvement of Oxidative Stress and Poly (ADP-ribose) Polymerase Activation in 3-Nitropropionic Acid-induced Cytotoxicity in Human Neuroblastoma Cells

3-Nitropropionic acid (3-NP) inhibits electron transport in mitochondria, leading to a metabolic failure. In order to elucidate the mechanism underlying this toxicity, we examined a few biochemical changes possibly involved in the process, such as metabolic inhibition, generation of reactive oxygen species (ROS), DNA strand breakage, and activation of Poly (ADP-ribose) polymerase (PARP). Exposure of SK-N-BE (2) C neuroblastoma cells to 3-NP for 48 h caused actual cell death, while inhibition of mitochondrial function was readily observed when exposed for 24 h to low concentrations (0.2~2 mM) of 3-NP. The earliest biochemical change detected with low concentration of 3-NP was an accumulation of ROS (4 h after 3-NP exposure) followed by degradation of DNA. PARP activation by damaged DNA was also detectable, but at a later time. The accumulation of ROS and DNA strand breakage were suppressed by the addition of glutathione or N-acetyl-L-cysteine (NAC), which also partially restored mitochondrial function and cell viability. In addition, inhibition of PARP also reduced the 3-NP-induced DNA strand breakage and cytotoxicity. These results suggest that oxidative stress and activation of PARP are the major factors in 3-NP-induced cytotoxicity, and that the inhibition of these factors may be useful in protecting neuroblastoma cells from 3-NP-induced toxicity.

Ceramide is Involved in MPP+ -induced Cytotoxicity in Human Neuroblastoma Cells

To understand the cytotoxic mechanism of MPP+, we examined the involvement of ceramide in MPP+ -induced cytotoxicity to human neuroblastoma SH-SY5Y cells. When SH-SY5Y cells were exposed to MPP+, MPP+ induced dose-dependent cytotoxicity accompanied by 2-fold elevation of intracellular ceramide levels in SH-SY5Y cells. Three methods were used to test the hypothesis that the elevated intracellular ceramide is related to MPP+ -induced cytotoxicity: C2-ceramide was directly applied to cells, sphingomyelinase (SMase) was exogenously added, and oleoylethanolamine (OE) was used to inhibit degradation of ceramide. Furthermore, inhibition of ceramide-activated protein phosphatase (CAPP), the effector of ceramide, using okadaic acid (OA) attenuated cell death but treatment of fumonisin B1, the ceramide synthase inhibitor, did not alter the cytotoxic effect of MPP+. Based on these, we suggest that the elevation of intracellular ceramide is one of the important mediators in MPP+ -induced cell death.

Study of dopamine transporter in the rat 6-hydroxydopamine Parkinson's disease model

BACKGROUND: Neuroprotective therapy is essential in the management of Parkinson's disease(PD). As symptomatic benefit of a treatment may clinically mask the disease progression, an evaluation of the effect of a neuroprotective therapy should be based on objective measurement of in vivo dopaminergic integrity: Nuclear imaging techniques such as SPECT or PET can visualize dopaminergic system using dopamine transporter ligands and show the promise for this purpose. The objective of this study is to examine the changes of dopamine transporter in the animal model of PD and those correlations with behavioral and biochemical changes. METHODS: We injected 6-hydroxydopamine into the substantia nigra in Sprague-Dawley rats to establish the unilateral PD model, and examined the rotation response after apomorphine injection as a behavioral aspect of the animal model. And we also measured the dopamine and DOPAC level in the striata and the dopamine transporter by [3H]-mazindol autoradiography. RESULTS: We observed that the rats showed turning behavior only after severe reduction of dopamine and DOPAC. There was a strong inverse correlation between rotation behavior and striatal dopamine, DOPAC and dopamine transporter density. There was a positive and strong linear-correlation between dopamine transporter density and dopamine or DOPAC levels. CONCLUSION: Measurement of dopamine transporter gives a good estimate of striatal dopamine level in an animal model of PD. In vivo measurement of dopamine transporter will give an objective information on the integrity of presynaptic nigrostriatal dopaminergic system.