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

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CHAPTER 3 MATERIALS AND METHODS Chemicals 122 Aluminium chloride hexahydrate, BSA, CAT, cytochrome c, desipramine hydrochoride, 3,3‟-diaminobenzidine, 2,4-dinitrophenylhydrazine hydrochloride, 5,5‟- dithiobis-(2-nitrobenzoic acid), EDTA, GPx, GR, H2O2, ketamine/xylazine, mouse monoclonal antibody to TH, TBA, xanthine, and xanthine oxidase were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Avidin-biotin-peroxidase complex and biotinylated secondary antibody were purchased from Vector (Burlingame, CA, USA). The water used for the preparations of solutions was Milli-RiOs/Q-A10 grade, (Millipore Corp., Bedford, MA, USA). All remaining chemicals used were of analytical grade and were purchased from Fluka Chemie AG (Buchs, Switzerland). Animal treatment Adult male Sprague-Dawley rats (200-250 g) were used to perform the in vivo studies. All animals were housed individually in polypropylene cages to reduce extraneous trace element contamination in a room equipped with 12 h light/dark automatic light cycles, maintained at 22�1°C with a relative humidity of 65%. All experiments were carried out in accordance with the “Principles of laboratory animal care” (NIH publication No. 86-23, revised 1996) and approved by the corresponding committee at the University of Santiago de Compostela. Aluminium dosage was adjusted according to animal‟s body weight just before each experiment. All rats were allowed a standard maintenance diet (A04, Panlab S.L., Barcelona, Spain) and water ad libitum. Animals were randomly assigned to two experimental groups. The first group was subdivided into two subgroups, each consisting of ten animals: rats in subgroup A were daily i.p. injected with aluminium chloride in saline (NaCl 0.9%) at a dose of 10 mg Al 3+ /kg for 10 days; rats in subgroup B were injected with the same volume of
CHAPTER 3 saline over the same period. The second group was subdivided into four subgroups of 11 animals each: rats in subgroup C were used as normal (i.e. non-lesioned) controls, and received the corresponding injections of vehicle for 10 days; rats in subgroup D were i.p. injected with aluminium chloride at a dose of 10 mg Al 3+ /kg/day for 10 consecutive days; rats in subgroup E received i.p. injections of saline for 10 days and were lesioned on the 8th day (two hours after i.p. injection) with 200 µg 6-OHDA in 3 µl sterile saline containing 0.2% ascorbic acid injected in the third ventricle; rats in subgroup F were i.p. injected with aluminium chloride (10 mg Al 3+ /kg/day) for 10 days and were lesioned with 6-OHDA in the same way as subgroup E. Injection solutions containing aluminium were prepared by dissolving aluminium chloride in saline, adjusting to pH 4.6 with sodium hydroxide, and waiting the equilibration time necessary to obtain a clear solution. In all cases the volume injected was 0.5 ml. Stereotaxic coordinates for intraventricular injection of 6-OHDA were 0.8 mm posterior to bregma, midline, 6.5 mm ventral to the dura, and tooth bar at 0. The solution was injected with a 10 µl Hamilton syringe coupled to a motorized injector (Stoelting, Wood Dale, IL, USA), at 0.5 µl/min, and the cannula was left in situ for 5 min after injection. All surgery was performed under ketamine/xylazine anesthesia, and 30 min prior to injection of 6-OHDA, rats received desipramine (25 mg/kg i.p.) to prevent uptake of 6-OHDA by noradrenergic terminals. The accuracy of the lesions and cannula placement were confirmed by post-mortem analysis with cresyl violet staining. Brain samples At the end of the experimental period animals of subgroups A and B were stunned with carbon dioxide and sacrificed by decapitation. Brains were quickly removed and rinsed with ice-cold saline. Regional brain segments (cerebellum, ventral midbrain, cortex, hippocampus, striatum) according to Paxinos and Watson (2007) were immediately dissected out on an ice plate. For biochemical assays, weighed brain samples were individually homogenized in a Na2PO4/KH2PO4 buffer (pH 7.4) isotonized with KCl (1:6 w/v for cerebellum, 1:2 for cortex; 1:30 for ventral midbrain; 123
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Universidade de Santiago de Compost
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Don Ramón Soto Otero y Doña Estef
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Acknowledgements The work of this t
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Abbreviations AA ascorbic acid AD A
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Abbreviations (continuation) PCC pr
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List of figures (continuation) Chap
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TABLE OF CONTENTS INTRODUCTION ....
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CHAPTER 3 Brain oxidative stress an
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INTRODUCTION PARKINSON’S DISEASE
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INTRODUCTION PD is found in all eth
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Bradykinesia INTRODUCTION Bradykine
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INTRODUCTION predate the occurrence
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Table 1: Differential diagnostic in
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INTRODUCTION Table 3: National Inst
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Figure 4: Dopamine catabolism (Mén
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The basal ganglia INTRODUCTION The
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The death of SNpc DAergic neurons I
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INTRODUCTION the dorsomedial part o
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INTRODUCTION characterized as are l
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INTRODUCTION (Olanow et al. 2004).
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Etiology and pathogenesis of PD INT
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Ageing INTRODUCTION Ageing remains
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INTRODUCTION The finding of MPTP to
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INTRODUCTION may clarify why many n
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Misfolding and aggregation of prote
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INTRODUCTION (E3) (Figure 18A). Los
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INTRODUCTION in connection with Par
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INTRODUCTION Oxidative damage happe
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DA metabolism as a source of ROS IN
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Contribution of oxidative and nitro
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Excitotoxicity INTRODUCTION Glutama
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INTRODUCTION al. 1996, Cicchetti et
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Experimental models of PD INTRODUCT
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INTRODUCTION induce selective DAerg
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ALUMINIUM General features INTRODUC
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INTRODUCTION Varying amounts of alu
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INTRODUCTION and antiperspirants co
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INTRODUCTION approximately 3% of al
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Excretion INTRODUCTION As insoluble
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INTRODUCTION 3.5 mg may be increase
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INTRODUCTION Oteiza 1999), non-iron
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Aluminium as a cell membrane menace
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INTRODUCTION mobilization of Ca 2+
Page 95: Aluminium impairs neurotransmission
Page 99 and 100: AIMS OF THE PRESENT THESIS The main
Page 101: OBJETIVOS
Page 104 and 105: OBJETIVOS 3) Evaluar de forma preci
Page 107: CHAPTER 1 Time-course of brain oxid
Page 110 and 111: CHAPTER 1 INTRODUCTION 86 6-OHDA (2
Page 112 and 113: CHAPTER 1 MATERIALS AND METHODS Che
Page 114 and 115: CHAPTER 1 followed by centrifugatio
Page 116 and 117: CHAPTER 1 RESULTS Effects of 6-OHDA
Page 118 and 119: CHAPTER 1 DISCUSSION 94 As shown by
Page 120 and 121: CHAPTER 1 strategies or to study th
Page 122 and 123: CHAPTER 1 Fig. 2 Changes in PCC in
Page 125: CHAPTER 2
Page 129 and 130: ABSTRACT CHAPTER 2 In the present w
Page 131 and 132: MATERIALS AND METHODS Chemicals CHA
Page 133 and 134: CHAPTER 2 atomization used were 1,5
Page 135 and 136: DISCUSSION CHAPTER 2 Aluminium ente
Page 137: Table 1. Graphite furnace programme
Page 141: CHAPTER 3 Brain oxidative stress an
Page 144 and 145: CHAPTER 3 INTRODUCTION 120 The huma
Page 148 and 149: CHAPTER 3 1:15 for hippocampus and
Page 150 and 151: CHAPTER 3 initially incorporated to
Page 152 and 153: CHAPTER 3 RESULTS In vivo effects o
Page 154 and 155: CHAPTER 3 Effects of aluminium admi
Page 156 and 157: CHAPTER 3 DISCUSSION 132 Aluminium
Page 158 and 159: CHAPTER 3 hippocampus, showed simil
Page 160 and 161: CHAPTER 3 assume that the distinct
Page 162 and 163: CHAPTER 3 Fig. 1 Levels of TBARS (A
Page 164 and 165: CHAPTER 3 Fig. 2 Enzyme activity of
Page 166 and 167: CHAPTER 3 Fig. 3 Microphotographs s
Page 168 and 169: CHAPTER 3 Fig. 5 Levels of TBARS (A
Page 170 and 171: CHAPTER 3 Fig. 6 In vitro effects o
Page 173: SUMMARY
Page 176 and 177: SUMMARY values were attained at 48
Page 178 and 179: SUMMARY neurodegeneration in the DA
Page 181 and 182: CONCLUSIONS The results obtained in
Page 183: 13) Aluminium does affect neither M
Page 187 and 188: RESUMEN RESUMEN Desde el punto de v
Page 189 and 190: RESUMEN zonas cerebrales excepto en
Page 191: CONCLUSIONES
Page 194 and 195: CONCLUSIONES Capítulo 2 4) La admi
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CONCLUSIONES 172 En base a las conc
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Publications as a direct result fro
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