The present study demonstrates for the first time that MB exerts protective effects against structural, neurochemical and behavioral deficits induced by the direct intrastriatal infusion of Rot in rats. The anatomopathologic features of the damage produced by the intrastriatal injection of Rot observed in this study (i.e., liquefactive necrosis and reactive gliosis) are akin to those peculiar to the lesions described in patients with certain types of inborn errors of metabolism (Heidenreich et al., 1988
, Roodhooft et al., 1990
). These lesions have been reported to be a form of neurotoxic degeneration independent from hypoxemia and/or vascular deficiency. Rather they stem from the neurotoxic effects elicited by accumulating organic acid metabolites, which eventually lead to mitochondrial dysfunction (Brismar and Ozand, 1994
, Gascon et al., 1994
, Okun et al., 2002
). Such lesions consist of foci of necrosis, gliosis and spongiosis that appear as bilateral hypodensities, T2
hyperintensities and atrophy of the striatum, putamen or globus pallidus (Korf et al., 1986
, Brismar and Ozand, 1994
, Gascon et al., 1994
). The current study presents evidence supporting that similar degenerative changes can be induced by the infusion of the neurotoxin Rot.
Rot has been classically characterized as a selective inhibitor of mitochondrial complex I (Palmer et al., 1968
, Gutman et al., 1970
, Greenamyre et al., 2001
). Its neurotoxic mechanism of action in striatal cells has been shown to be related to production of reactive oxygen species (Moldzio et al., 2008
). Complex I has been regarded as a major source of reactive oxygen species, especially when its function is inhibited by Rot (Lenaz et al., 2006
). In line with this, neural cells transfected with Rot-insensitive single-subunit complex I do not display mitochondrial impairment, oxidative damage, or death (Sherer et al., 2003
), which supports that Rot-induced increases in oxidative stress are secondary to complex I inhibition. Nevertheless, dopaminergic cells from knockout mice deficient in functional complex I showed no increases in cell death, and in fact were more sensitive to Rot toxicity (Choi et al., 2008
). Hence, although Rot might induce neurodegeneration via
inhibition of complex I with a concomitant increase in oxidative stress, alternate neurotoxic mechanisms and sources of oxidative stress secondary to Rot exposure seem to be possible. Rot-induced oxidative damage lowers the threshold for activation of mitochondrial-dependent apoptosis and makes compromised neurons more likely to degenerate (Perier et al., 2005
). In this study we provide evidence that both increases in oxidative stress and impairment in mitochondrial respiration are associated with Rot-induced striatal damage. Similar to what has been previously shown with whole brain mitochondria (Rojas et al., 2009
), isolated striatal mitochondria incubated with Rot manifested an immediate compromise of complex I activity. Rot also induced early decrease in striatal energy metabolism capacity in vivo
and induced increases in perilesional striatal superoxide levels.
A first clear effect of MB observed in the present study was its ability to visibly reduce the extent of Rot-induced striatal degeneration, an effect that was evident in the bilateral and unilateral models of Rot-induced damage. Co-administration of MB displayed a series of metabolic effects that were not restricted to the core of the Rot-induced lesion, but were extended to the lesion penumbra, the contralateral striatum and distant motor regions. At the lesion core, MB prevented the decrease in cytochrome oxidase activity induced by Rot, supporting a metabolic enhancing effect. On the other hand, MB actually showed a paradoxical decreased in cytochrome oxidase activity in the lesion penumbra. The penumbra displays peculiar neurochemical conditions including impaired electric conduction, glutamate receptor hyperactivation, calcium overload, increased oxidative stress and damage to the neurovascular matrix, that render it different from the surrounding spared tissue (Lo, 2008
). We observed increases in penumbral oxidative stress in the Rot group that were decreased in the Rot/MB group, evidencing the antioxidant effects of MB. The combined effects of MB on perilesional oxidative stress and cytochrome oxidase activity support a mechanism in which an antioxidant effect parallels decreases in excitotoxicity that underlie prevention of structural damage.
In in vitro
experiments with striatal mitochondria, MB treatment showed no effect on Rot-induced inhibition of complex I activity, and there were no between-group differences in in situ
striatal complex I activity. These results suggest that MB’s neuroprotection in the striatum is likely not mediated by direct enhancement of complex I activity. The data also rule out the possible molecular interaction of MB with Rot as a mechanism mediating neuroprotection. In addition, chromatographic analyses have shown that Rot and MB do not interact chemically when both are present in the same solution (Zhang et al., 2006
). However, we found that MB boosted ipsilateral striatal metabolic capacity as measured with cytochrome oxidase activity, compared to the contralateral striatum. MB studies in our laboratory were the first to show that MB increases brain oxygen consumption (Riha et al., 2005
, Zhang et al., 2006
) and brain cytochrome oxidase activity both in vitro
and in vivo
(Callaway et al., 2004
, Gonzalez-Lima and Bruchey, 2004
, Wrubel et al., 2007
), and improves brain function in vivo
in the presence of sodium azide, a cytochrome oxidase inhibitor (Callaway et al., 2002
). Atamna et al. (2008)
also showed in fibroblasts in tissue culture that MB delays cell senescence by increasing oxygen consumption and preventing the formation of oxidants through cycling of MB between oxidized and reduced forms. Therefore, based on the above observations, it can be hypothesized that the antioxidant and metabolic effects of MB could impact neuronal survival.
Of relevance to the understanding of the potential protective effects of MB on brain function under toxic conditions is the finding that the modulation of metabolic activity by MB was not restricted to the lesion and its vicinity. Conversely, a decreased metabolic activity in the contralateral striatum was observed in the Rot/MB group but not in the Rot group, a finding that supports the ability of MB co-administration in counteracting contralateral compensatory responses produced by Rot-induced lesions. Furthermore, MB co-administration was found to influence the metabolic activity in both close and remote structures of a neural network involved in motor control. Indeed, it was observed that the presence of a striatal lesion induced a marked ipsilateral activation of basal ganglia regions, thalamus and cortex in both the Rot- and Rot/MB-treated subjects. However, the patterns of combined activation in both groups were different. Thus, brains in the Rot group featured a functionally decoupled ipsilateral motor network, an effect that was prevented in Rot/MB brains, as demonstrated by the high interregional correlations observed in this group. To correctly interpret the previous data, it is important to highlight that the reported interregional correlations do not depend on the type of online and task-dependent regional activation, as observed with other functional neuroimaging modalities like fMRI or PET. Instead, the metabolic mapping method based on cytochrome oxidase activity likely reflects long-lasting changes in brain metabolic capacity determined by sustained energy demands (Sakata et al., 2000
, Sakata et al., 2005
). Therefore, the observed between-group differences in regional brain metabolism provide evidence that MB co-administration counteracts the neurotoxicity of Rot not only by limiting its deleterious effects on brain structure but also by triggering potentially advantageous functional network changes in brain metabolism, which may contribute to adaptive rewiring (Dancause et al., 2005
The protective effects of MB against Rot-induced striatal damage were also observed at the behavioral level. A beneficial effect of MB against Rot-induced behavioral impairment was observed in the dot removal test where MB co-administration attenuated the bias in the removal of the contralateral dot, as well as the decrease in latency to contralateral dot contact observed in Rot-treated rats. Similarly, the vibrissae stimulation-evoked placing test also revealed within-group score decrements in Rot-treated rats that were not observed in Rot/MB-treated rats. Thus these two behavioral tests showed sensitivity not only to the onset and severity of motor asymmetries, but also to differences between highly impaired subjects (i.e. in the Rot group) and mildly impaired subjects (i.e. in the Rot/MB group).
The findings are consistent with MB’s protection against Rot neurotoxicity found in the retina previously (Zhang et al., 2006
, Rojas et al., 2009
) and further support the possibility that MB may exert its neuroprotective effects in vivo
by means of a dual molecular mechanism implicating both antioxidant and metabolic enhancing effects on mitochondrial respiration. Given that Rot-induced neurodegeneration is particularly contingent on increases of oxidative stress as opposed to bioenergetic failure alone (Sherer et al., 2003
), the tissue sparing effects of MB can be attributed mainly to its powerful antioxidant effects. Indeed, imino-containing compounds such as MB are known to have superior antioxidant properties compared to vitamin E and phenolic compounds (Moosmann et al., 2001
). Nevertheless, MB’s unique chemical structure, featuring a central thiazine ring, allows it to be not only very susceptible to reduction, but at the same time easily auto-oxidized, depending on MB’s concentration and the redox conditions of the milieu. In vivo
, at low MB concentrations such as used in this study, oxidized and reduced forms of MB are at equilibrium, and function as a reversible redox system (Bruchey and Gonzalez-Lima, 2008
) with powerful antioxidant and respiratory enhancing effects (Zhang et al., 2006
). Thus, MB can enter a cycle of oxidation and reduction which eventually can prevent oxidative damage and sustain mitochondrial function. These respiratory enhancing effects of MB appear related to the generalized brain network and behavioral effects detected in this study.
The present findings support that MB could be a valuable intervention against neural damage associated with oxidative stress and energy hypometabolism. They also add to our previous work evidencing a possible therapeutic role of interventions targeting mitochondrial function in neurodegenerative disorders (Rojas et al., 2008a
, Pienaar et al., 2009
). It is important to notice, however, that the co-administration paradigm used in this study is limited in its capacity to reveal the potential neuroprotective value of MB after the onset of neurodegeneration or as an effective prophylactic intervention before neurodegeneration is evident. Thus, the effects of MB should be further investigated in paradigms that address these two conditions that are highly relevant for clinical applications. This implies a specific focus on the effects of pre-lesion and post-lesion as well as systemic MB administration.