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90%) of embryonic mesencephalic dopamine (DA) neurons die following transplantation to the striatum. Recent reports indicate that at least a subpopulation of grafted cells undergo apoptotic cell death at early times following implantation. This study examines the temporal pattern and magnitude of apoptotic cell death following the implantation of mesencephalic cell suspension grafts. Two techniques, a modified terminal deoxynucleotide-mediated nucleotide end labeling (TUNEL) technique and cresyl violet staining, are used to assess apoptotic cell death by detection of its biochemical and morphological identifiers, respectively. Male, Fischer 344 rats were examined at 1, 4, 7, and 28 days following implantation of embryonic day 14 (E14) ventral mesencephalic cells to the DA-denervated striatum. Results indicate that the overwhelming majority of apoptotic cell death occurs within the first 7 days after transplantation. However, the impact of the apoptosis that occurs over the first week following grafting only appears to limit grafted tyrosine hydroxylase-immunoreactive (THir) neuron survival during the first 4 days. No significant differences between the survival rates of THir neurons at 4 days after grafting and at 28 days after grafting were found. Therefore, it appears that the critical interval during which an estimated 90% of grafted DA neurons die is during the first 4 days postimplantation and that a major contributor to this cell death is apoptosis. 
doi:10.1016/j.fct.2006.04.002 
Copyright © 2006 Elsevier Ltd All rights reserved.
Quebrachitol (2-O-methyl-l-inositol) attenuates 6-hydroxydopamine-induced cytotoxicity in rat fetal mesencephalic cell cultures
H.V. Nobre Júniora, G.M.A. Cunhaa, M.O. Moraesa, M.F.D. Lucianaa, R.A. Oliveiraa, F.D. Maiaa, M.A.S. Nogueirab, T.L.G. Lemosb and V.S. Raoa, 
, 
Received 8 January 2006;
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Abstract
Naturally occurring plant substances have the potential to prevent oxidative damage in various pathophysiological conditions including neurodegenerative disorders. Recent findings indicate that impaired energy metabolism plays a prominent role in neurodegeneration. The present study investigated whether quebrachitol (2-O-methyl-l-inositol) (QCT), a sugar like natural compound that was suggested to have both antioxidant and membrane stabilization activity prevents the cytotoxic effect of 6-hydroxydopamine (6-OHDA, 200 μM) on cultured rat fetal mesencephalic cells. While QCT (0.1–100 μg/ml) produced no effect per se on cell viability as measured in the 3[4,5-dimethylthiazole-2il]-2,5-diphenyltetrazolium bromide (MTT) test, it offered concentration-related protection against cell death induced by 6-OHDA. In addition, QCT demonstrated an antioxidant activity against 6-OHDA-induced oxidative stress as evidenced by reduced formation of nitrite–nitrate and thiobarbituric acid-related substances. Fluorescence microscopy using acridine orange/ethidium bromide double staining further affirmed the absence of 6-OHDA (200 μM)-induced morphological changes characteristic of apoptosis/necrosis in cultures pretreated with QCT (100 μg/ml). Also, results of tyrosine hydroxylase immunoreactivity indicated that 6-OHDA induces cell death in mesencephalic cultures affecting both TH+ positive and TH− negative (TH+ and TH−, respectively) and QCT pretreatment protects them from cell death, in a non-specific manner. Our data indicate that QCT has a cytoprotective role due, at least in part, to an antioxidant and free radical scavenging mechanism. Furthermore, the study suggests that inositol compounds might serve as leads in developing drugs for the treatment of various neurodegenerative disorders.
Keywords: Quebrachitol; 2-O-Methyl-l-inositol; 6-Hydroxydopamine; Cytotoxicity; Mesencephalic cell cultures; Fetal rat
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Plant material and isolation of QCT (2-O-methyl-l-inositol)
- 2.2. Chemicals
- 2.3. Animals
- 2.4. Mesencephalic cell cultures
- 2.5. Culture treatments
- 2.6. Cell viability (MTT assay)
- 2.7. Analysis of cell death by fluorescence microscopy
- 2.8. Tyrosine hydroxylase immunoreactivity
- 2.9. Nitrite–nitrate determination
- 2.10. Lipid peroxidation assay
- 2.11. Statistical analysis
- 3. Results
- 3.1. Effect on cell viability
- 3.2. Fluorescence microscopy findings
- 3.3. Tyrosine hydroxylase immunoreactivity
- 3.4. Effect on the release of NO
- 3.5. Effect on lipid peroxidation
- 4. Discussion
- Acknowledgements
- References
