Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...

CHAPTER 3
1:15 for hippocampus and 1:10 for striatum) in a Dounce tissue grinder (Kontes,
Vineland, NJ, USA). The homogenates were then centrifuged at 14,000 g for 20
minutes at 4°C. The supernatants were aliquoted and stored at –80°C to determine
protein content and the activity of GPx, SOD, and CAT. The resulting pellets were
resuspended with cold Na2PO4/KH2PO4 buffer (pH 7.4) isotonized with KCl and
containing 20 �M BHT and 20 �M desferrioxamine. These compounds were used to
prevent amplification of lipid peroxidation during the progression of the analysis. Then,
homogenates were immediately sonicated for 5 seconds (250 Digital Sonifer, Branson
Ultrasonic Co., Danbury, CT, USA). These samples were used for the quantification of
TBARS, PCC, PTC, and protein concentration.
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Forty-eight hours after lesion with 6-OHDA, five rats of subgroups C, D, E, and
F were killed according to the above cited procedure in order to perform biochemical
studies because it has been previously reported that the peak for oxidative stress is
reached at this time (Sánchez-Iglesias et al. 2007a). In these rats, the levels of the
oxidative stress caused by 6-OHDA were estimated by quantification of both lipid
peroxidation and protein oxidation in ventral midbrain and striatum.
Preparation of brain mitochondria
Brain mitochondria from Sprague-Dawley rats weighing 200-250 g were
obtained by differential centrifugation according to a previously published method
(Méndez-Álvarez et al. 1997) and protein concentration determined according to
Markwell et al. (1978), using BSA as the standard.
Determination of TBARS, PCC, and PTC
The TBARS, PCC, and PTC determinations for in vitro and in vivo experiments
were performed spectrophotometrically as previously described in Hermida-Ameijeiras
et al. (2004) and Sánchez-Iglesias et al. (2007a), respectively. 2,4-
Dinitrophenylhydrazine hydrochloride and 5,5‟-dithiobis-(2-nitrobenzoic acid) were