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

CHAPTER 3
initially incorporated to the reaction mixture, followed by 100 �l of GPx (0.2 U) instead
of brain sample.
Measurement of CAT activity
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CAT activity was polarographically measured following the formation of O2
using a Clark-type oxygen electrode, according to a slight modification to a previous
published method (Méndez-Álvarez et al. 1998). Briefly, 100 �l brain sample were
incubated in the electrode chamber and the reaction started with the addition of 20 �l of
a 500 �M solution of H2O2. CAT activity was expressed as U/mg protein. For in vitro
studies, 100 �l of CAT (20 U) were first added to the reaction mixture, followed by
different concentrations of Al 3+ (10, 50, 100 �M) in order to estimate the effect of the
metal on CAT activity.
Determination of MAO activity
MAO activity was spectrophotometrically measured in mitochondria
preparations as previously described (Soto-Otero et al. 2001), using kynuramine as a
non-selective substrate and (-)-deprenyl and clorgyline as irreversible inhibitors for
MAO-A and MAO-B estimation, respectively. Different concentrations of Al 3+ (10, 50,
100 �M) were incorporated to the incubation in order to assess the effect of aluminium
on both MAO-A and MAO-B activity.
Immunohistochemistry
The remaining six rats of subgroups C, D, E, and F were processed for
immunohistochemistry. Animals were killed by a chloral hydrate overdose one week
post-lesion and then processed for TH immunohistochemistry according to a previously
published methodology (Rey et al. 2007).