Overhauser-enhanced magnetic resonance imaging characterization of mitochondria functional changes in the 6-hydroxydopamine rat model
Highlights
► Using OMRI, we noninvasively demonstrated that brain redox alterations occur in 6-OHDA-lesioned rats. ► Redox alterations were reflected in changes in mitochondrial complex I activity. ► Redox imaging may be useful for noninvasively evaluating the progress of Parkinson’s disease.
Introduction
Parkinson’s disease is a progressive, neurodegenerative condition characterized by deterioration of the dopaminergic neurons of the substantia nigra pars compacta (Lees et al., 2009). The exact mechanisms by which neuronal cell death in Parkinson’s disease occurs remain unknown, but several lines of evidence suggest that mitochondrial dysfunction induced by oxidative stress may play an important role. Mitochondrial dysfunctions, including complex I deficiencies and impaired electron transfers, have been reported in Parkinsonian patients (Mizuno et al., 1989, Schapira et al., 1989) and animal models (Dabbeni-Sala et al., 2001, She et al., 2011).
Recently, the development of novel imaging techniques has permitted the noninvasive in vivo study of neurodegenerative processes. The 6-hydroxydopamine (6-OHDA) model in rats is widely used to study the mechanisms involved in the natural course of Parkinson’s disease (Breese et al., 1984, Mendez and Finn, 1975). Fox example, following administration of 6-OHDA, T2-weighted magnetic resonance imaging (MRI) showed hyperintensities in the area in which the 6-OHDA was injected, and these hyperintensities were accompanied by reductions in the number of tyrosine hydroxylase-positive cells (Kondoh et al., 2005). In addition, blood oxygenation level-dependent functional MRI demonstrated bilateral alterations in the cortical and striatal activity of 6-OHDA-lesioned rats (Pelled et al., 2005). These findings suggest that imaging techniques may be highly useful for evaluating neurological damage in vivo. However, although the progress of Parkinson’s disease has been associated with mitochondrial dysfunction when measured by invasive means, less success has been achieved using noninvasive imaging techniques.
During the mitochondrial respiratory chain reaction, electrons are transferred from electron donors to electron acceptors, such as oxygen, through redox reactions. To detect the redox status of mitochondria, nitroxyl radicals (i.e., nitroxides) may be effective redox-sensitive spin probes. Nitroxyl radicals can be reduced enzymatically by complex I and complex II in the electron transport chain of mitochondria (Chen et al., 1988, Quintanilha and Packer, 1977). These reactions form the chemical basis for using nitroxyl radicals as contrast agents for Overhauser-enhanced MRI (OMRI) to determine mitochondrial function in the brain (Yamato et al., 2009). OMRI is a double resonance technique that uses the presence of paramagnetic agents to enhance the signal intensity from nuclear spins by a process known as dynamic nuclear polarization, or the Overhauser effect (Krishna et al., 2002, Li et al., 2006, Lurie et al., 1988).
The aim of the present study was to investigate functional features of mitochondrial dysfunction in the 6-OHDA-lesioned experimental model using OMRI. In this regard, we used 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-l-oxyl (methoxycarbonyl-PROXYL) as a redox-sensitive contrast agent, because it has good permeability through the blood-brain barrier, as confirmed by MRI (Hyodo et al., 2008), autoradiography (Anzai et al., 2003), and microdialysis studies (Shiba et al., 2008). To identify the mechanisms responsible for the reduction of methoxycarbonyl-PROXYL in the brain redox image, the signal reduction rates in the cellular fractions were estimated by X-band ESR. For a comprehensive investigation of mitochondrial dysfunction, we also biochemically examined the activity of the mitochondrial electron transport chain enzymes. In addition, we performed spin-trapping experiments to clarify whether 6-OHDA directly induced free radical generation, which has been associated with mitochondrial dysfunction.
Section snippets
Chemicals
3-Carboxy-2,2,5,5-tetramethylpyrrolidine-l-oxyl (carboxy-PROXYL) was purchased from the Aldrich Chemical Co. (Milwaukee, WI, USA). NADH was purchased from the Oriental Yeast Co., Ltd. (Tokyo, Japan). 6-Hydroxydopamine hydrochloride (6-OHDA) and decylubiquinone were purchased from the Sigma–Aldrich Co. (St. Louis, MO, USA). Antimycin A was purchased from MP Biomedicals, LLC. (CA, USA). 2,6-Dichlorophenolindophenol was purchased from the Katayama Chemical, Inc. (Osaka, Japan).
Behavioral dysfunction and a loss of dopamine in 6-OHDA-lesioned rats
We confirmed the presence of behavioral dysfunctions and dopamine depletions in 6-OHDA-lesioned rats, a model of Parkinson’s disease. In rats unilaterally administered 6-OHDA, 6 weeks after the microinjection, administration of methamphetamine induced movements towards the side of the body ipsilateral to the site of the 6-OHDA lesion (Fig. 1a). In addition, dopamine levels in the striatum were significantly lower in the lesioned hemisphere (p < 0.01, Fig. 1b).
Redox imaging using OMRI
Fig. 2a shows tubes containing the
Discussion
The results of this study show that reduction of methoxycarbonyl-PROXYL was influenced by 6-OHDA lesions. The reduction of nitroxyl radicals is a useful index for estimating the mitochondrial redox status noninvasively, using OMRI. Our findings suggest that redox imaging of the brain reflects alterations in complex I activity in mitochondria. Spin trapping also demonstrated that 6-OHDA directly induced the generation of the hydroxyl radicals associated with mitochondrial dysfunction.
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Both these authors contributed equally to this work.