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

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CHAPTER 2 experimental period of four weeks. Aluminium dosage was adjusted according to the animal‟s body weight just before experiment. All rats received a standard diet (A04, Panlab, Barcelona, Spain) and the corresponding drinking (saline or aluminium salt+citric acid) ad libitum. Body weight and fluid intake were measured three times a week to adjust the doses in order to achieve a constant aluminium and citric acid intake. Brain samples 108 After treatment, animals were sacrificed by decapitation and brains quickly excised. Regional brain segments [cerebellum (CE), ventral midbrain (VM), cortex (CO), hippocampus (H), striatum (ST)] were rapidly dissected out on ice plate, according to Paxinos and Watson (2007). Weighed brain regions were stored at –40°C until trace element analysis. Electrothermal atomic absorption spectrometry (ETAAS) The samples were homogenised and digested with nitric acid (HNO3) and H2O2, using microwave energy. A portion of 500 mg of sample was weighed into a digestion vessel and 2 mL of HNO3 were added. Afterwards, vessels were introduced into the microwave oven at 100 W during 12 min. Then, 1 mL of H2O2 was added into digestion vessel and introduced into the microwave oven during 10 min at 300 W. Subsequently, the sample was adjusted to its final volume (5 mL) by addition of ultrapure water. A portion of 20 µL of the digested sample was introduced into the graphite furnace tube for its analysis by ETAAS. Measurements were performed using an atomic absorption spectrometer Model 1100 B (Perkin-Elmer, Norwalk, CT, USA), equipped with an HGA-700 graphite furnace atomizer and AS-70 autosampler. A hollow cathode lamp operating at 25 mA, which provided a 309 nm line with a spectral bandwidth of 0.7 nm, was used. Deuterium background correction and pyrolytic graphite coated tubes with L´vov platforms were employed. All measurements made during this study used integrated absorbance with an integration time of 5 s. The mineralization and
CHAPTER 2 atomization used were 1,500 and 2,800 ºC respectively (Table 1). The volume injected was 20 µL. Each sample was digested by triplicate and measured by duplicate. Aluminium concentrations in samples were calculated using a standard addition method in an analytical range of 0-20 µg/L. The limit of detection was 0.12 µg/g. The accuracy of the method was investigated using the Reference Material DORM-2 with a certificated aluminium concentration of 10.9±1.7 µg/g, being the aluminium concentration obtained 11.4±2.2 µg/g. Reagents for aluminium determination were of the highest available purity in order to avoid any risk of sample contamination. All glassware was washed and kept in 10% (v/v) HNO3 for at least 48 hours and then rinsed with ultrapure water (Milli-RiOs/Q-A10). Statistical analysis Results were expressed as the mean ± SD. Statistical differences were tested using one-way ANOVA followed by Bonferroni‟s test for multiple comparisons. The accepted level of statistical significance was p < 0.05. All statistical procedures were carried out using the Statgraphics Plus 5.0 statistical package (Manugistics, Rockville, MD, USA). 109
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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
Page 81 and 82: INTRODUCTION and antiperspirants co
Page 83 and 84: INTRODUCTION approximately 3% of al
Page 85 and 86: Excretion INTRODUCTION As insoluble
Page 87 and 88: INTRODUCTION 3.5 mg may be increase
Page 89 and 90: INTRODUCTION Oteiza 1999), non-iron
Page 91 and 92: Aluminium as a cell membrane menace
Page 93 and 94: 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: MATERIALS AND METHODS Chemicals CHA
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 146 and 147: CHAPTER 3 MATERIALS AND METHODS Che
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
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13) Aluminium does affect neither M
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RESUMEN RESUMEN Desde el punto de v
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RESUMEN zonas cerebrales excepto en
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CONCLUSIONES
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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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