Mechanisms of aluminium neurotoxicity in oxidative stress-induced degenerative processes in relation to parkinson's disease

Resumen

Aluminium has become an important health concern due to both the frequent exposure to this metal and the suggested ability of aluminium to cause neurodegeneration. Pathological conditions such as PD have been associated with the accumulation of aluminium in brain. Although, aluminium is not a redox metal, it has been reported to be able to enhance brain oxidative stress. However, the molecular mechanism of its neurotoxicity remains not well understood. Consequently, we resolved to gain insight into the mechanisms of aluminium neurotoxicity in oxidative stress-induced degenerative processes in relation with PD. Before starting the whole procedures with aluminium, it was crucial to establish the kinetics of the oxidative damage induced in a 6-OHDA model of PD and also to quantify the changes observed in the indices of lipid peroxidation and oxidant status of proteins in striatum and ventral midbrain. These results would thereafter enable us to decide at which exact post-injection time should be performed the measurement of indices of brain oxidative stress when assessing the oxidative effects of aluminium. To accomplish the first part of this thesis, we chose the unilateral and intrastriatal injection of 6-OHDA to lesion the DAergic nigrostriatal pathway system in male Sprague-Dawley rats. This procedure has been extensively used to examine the degeneration of the DAergic neurons characteristics of PD. When compared to others 6-OHDA models with injections into the SN or the nigrostriatal tract, this method mimics more closely the progression of PD as it leads to a slower retrograde degeneration of the nigrostriatal system over a period of several weeks (Sauer and Oertel 1994, Przedborski et al. 1995, Lee et al. 1996, Shimohama et al. 2003). In fact, this allowed us to use different groups of rats sacrificed at distinct post-injection times (from 5 min to 7 days). Our results clearly indicated that unilateral and intrastriatal injection of 6-OHDA results in increased levels of lipid peroxidation and protein oxidation in the ventral midbrain and in the striatum. We observed for both areas an aument during the first 2 days post-injection and values returned to near control levels at the 7 day post-injection. Peak values were attained at 48 hours post-injection for both TBARS and PCC, and at 24 hours for PTC. Interestingly, lower but significant increases in oxidative stress levels were also seen in the contralateral side (ventral midbrain and striatum) excluding this zone as a control to determine neurochemical alterations caused by 6-OHDA in this experimental model of PD. At last and according to the obtained results, we decided to establish the optimal time of 48 hours post-injection for quantification of brain oxidative stress indices when assessing the oxidative potential of aluminium in this experimental model of PD. The second part of this thesis consisted in developing a dosage regimen of aluminium to rats which would guarantee a significant accumulation of this metal in brain areas and also to determine its precise distribution in the brain. As it was previously suggested that aluminium distribution depends on the animal species in question and the chemical form of aluminium administered, we opted for two distinct administration routes: oral and intraperitoneal. Sprague-Dawley rats were either daily i.p. injected with aluminium chloride (10 mg Al3+/kg/day) for 1 week, or given orally progressive increasing doses of aluminium chloride (25, 50, 100 mg Al3+/kg/day) supplemented with citrate (89, 178, 356 mg/kg/day) during 4 weeks. Our results showed that both administration routes led to aluminium accumulation. A greater and more significant increase was noted in the group receiving aluminium via intraperitoneal administration for most brain areas except in ventral midbrain. Distribution also varied with the administration route used. In accordance to these results we resolved to use the intraperitoneal administration route to clarify the brain oxidative stress provoked by aluminium. Finally the third phase of this thesis was dedicated to determine the ability of aluminium to alter the oxidant status of specific brain areas, such as cerebellum, ventral midbrain, cortex, hippocampus, and striatum. Male Sprague-Dawley rats were intraperitoneally administered with aluminium chloride (10 mg Al3+/kg/day) in saline for 10 days. As we demonstrated before, this dosage procedure was sufficient to insure aluminium accumulation in brain areas. Animals were sacrificed 48 hours after lesion to perform lipid peroxidation and protein oxidation studies because the peak for oxidative stress is reached at this time as we previously reported in the first phase of this thesis. Our results showed that, except for hippocampus, the metal triggered an increase in oxidative stress levels (determined as TBARS, PCC, and PTC) for most of the brain regions studied, which was accompanied by decreased activities of the antioxidant enzymes (SOD, CAT, and GPx). However, studies in vitro confirmed the inability of aluminium to affect the activity of those enzymes and also of MAO-A and MAO-B. The reported effects exhibited a regional-selective behaviour for all the cerebral structures studied. Worthy of note is the case of the hippocampus, as aluminium exposure resulted in increased antioxidant enzymes activities, no significant alterations of lipid peroxidation and decreased protein oxidations. These results might be explained by the high aluminium accumulation and the promotion of compensatory mechanism(s) in this brain area. Lastly, and to shed some light on the potential of aluminium to act as an etiological factor in PD, we studied the ability of this metal to increase the striatal DAergic neurodegeneration and various indices of oxidative stress in ventral midbrain and striatum of rats injected intraventricularly with 6-OHDA. This animal model was known to induce a more slowly ensuing parkinsonian syndrome exhibiting similar topographic depletion of DAergic neurons to that observed in PD (Rodriguez et al. 2001, Rodríguez-Díaz et al. 2001) and to avoid the focal lesion around the cannula tip produced when 6-OHDA is directly injected in the brain tissues. We followed the same dosage procedure as previously mentioned (daily intraperitoneal injection of 10 mg mg Al3+/kg/day in saline for ten days) except that rats were lesioned on the 8th day with 6-OHDA injected in the third ventricle. Animals were killed 1 week post-lesion for immunohistochemistry studies as it was reported that progressive degeneration of mesencephalic DAergic cells produced by intraventricular injection of 6-OHDA reached the definitive lesion pattern at the end of the first week postinjection (Rodriguez et al. 2001). Our results indicated that aluminium enhanced the ability of 6-OHDA to cause lipid peroxidation and protein oxidation (except for PTC in ventral midbrain). In addition, the metal was able to increase the capacity of 6-OHDA to cause neurodegeneration in the DAergic system as the loss of TH-ir striatal terminals was significantly increased. Note: It is also important to note that all experiments carried out during this thesis were in accordance with the Principles of laboratory animal care (NIH publication No. 86-23, revised 1996) and approved by the corresponding Ethics Committee at the University of Santiago de Compostela. Furthermore, all efforts were made to reduce the number of animals used and to minimize animal suffering.