Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...
Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...
Mechanisms of aluminium neurotoxicity in oxidative stress-induced ...
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CHAPTER 3<br />
sal<strong>in</strong>e over the same period. The second group was subdivided <strong>in</strong>to four subgroups <strong>of</strong><br />
11 animals each: rats <strong>in</strong> subgroup C were used as normal (i.e. non-lesioned) controls,<br />
and received the correspond<strong>in</strong>g <strong>in</strong>jections <strong>of</strong> vehicle for 10 days; rats <strong>in</strong> subgroup D<br />
were i.p. <strong>in</strong>jected with <strong>alum<strong>in</strong>ium</strong> chloride at a dose <strong>of</strong> 10 mg Al 3+ /kg/day for 10<br />
consecutive days; rats <strong>in</strong> subgroup E received i.p. <strong>in</strong>jections <strong>of</strong> sal<strong>in</strong>e for 10 days and<br />
were lesioned on the 8th day (two hours after i.p. <strong>in</strong>jection) with 200 µg 6-OHDA <strong>in</strong> 3<br />
µl sterile sal<strong>in</strong>e conta<strong>in</strong><strong>in</strong>g 0.2% ascorbic acid <strong>in</strong>jected <strong>in</strong> the third ventricle; rats <strong>in</strong><br />
subgroup F were i.p. <strong>in</strong>jected with <strong>alum<strong>in</strong>ium</strong> chloride (10 mg Al 3+ /kg/day) for 10 days<br />
and were lesioned with 6-OHDA <strong>in</strong> the same way as subgroup E. Injection solutions<br />
conta<strong>in</strong><strong>in</strong>g <strong>alum<strong>in</strong>ium</strong> were prepared by dissolv<strong>in</strong>g <strong>alum<strong>in</strong>ium</strong> chloride <strong>in</strong> sal<strong>in</strong>e,<br />
adjust<strong>in</strong>g to pH 4.6 with sodium hydroxide, and wait<strong>in</strong>g the equilibration time necessary<br />
to obta<strong>in</strong> a clear solution. In all cases the volume <strong>in</strong>jected was 0.5 ml.<br />
Stereotaxic coord<strong>in</strong>ates for <strong>in</strong>traventricular <strong>in</strong>jection <strong>of</strong> 6-OHDA were 0.8 mm<br />
posterior to bregma, midl<strong>in</strong>e, 6.5 mm ventral to the dura, and tooth bar at 0. The<br />
solution was <strong>in</strong>jected with a 10 µl Hamilton syr<strong>in</strong>ge coupled to a motorized <strong>in</strong>jector<br />
(Stoelt<strong>in</strong>g, Wood Dale, IL, USA), at 0.5 µl/m<strong>in</strong>, and the cannula was left <strong>in</strong> situ for 5<br />
m<strong>in</strong> after <strong>in</strong>jection. All surgery was performed under ketam<strong>in</strong>e/xylaz<strong>in</strong>e anesthesia, and<br />
30 m<strong>in</strong> prior to <strong>in</strong>jection <strong>of</strong> 6-OHDA, rats received desipram<strong>in</strong>e (25 mg/kg i.p.) to<br />
prevent uptake <strong>of</strong> 6-OHDA by noradrenergic term<strong>in</strong>als. The accuracy <strong>of</strong> the lesions and<br />
cannula placement were confirmed by post-mortem analysis with cresyl violet sta<strong>in</strong><strong>in</strong>g.<br />
Bra<strong>in</strong> samples<br />
At the end <strong>of</strong> the experimental period animals <strong>of</strong> subgroups A and B were<br />
stunned with carbon dioxide and sacrificed by decapitation. Bra<strong>in</strong>s were quickly<br />
removed and r<strong>in</strong>sed with ice-cold sal<strong>in</strong>e. Regional bra<strong>in</strong> segments (cerebellum, ventral<br />
midbra<strong>in</strong>, cortex, hippocampus, striatum) accord<strong>in</strong>g to Pax<strong>in</strong>os and Watson (2007) were<br />
immediately dissected out on an ice plate. For biochemical assays, weighed bra<strong>in</strong><br />
samples were <strong>in</strong>dividually homogenized <strong>in</strong> a Na2PO4/KH2PO4 buffer (pH 7.4)<br />
isotonized with KCl (1:6 w/v for cerebellum, 1:2 for cortex; 1:30 for ventral midbra<strong>in</strong>;<br />
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