Rotenone-induced nigrostriatal toxicity is reduced by methylene blue

This study investigated the effect of subcutaneously injected methylene blue on oxidative stress and nigrostriatal damage induced in the rat brain by systemic rotenone injection. Rotenone 1.5 mg/kg (injected subcutaneously three times per week) was given alone or in combination with methylene blue (5, 10, or 20 mg/kg subcutaneously daily) for 2 weeks. Brain concentrations of malondialdehyde, reduced glutathione, nitric oxide (nitrite), and acetylcholinesterase, paraoxonase 1 activity, and the antiapoptotic marker Bcl-2 (B-cell lymphoma 2) were determined. Histopathology, tyrosine hydroxylase immunoreactivity, tumor necrosis factor-alpha (TNF-α), and caspase-3 immunohistochemistry were also performed. Injection of rotenone resulted in increased oxidative stress in different regions of the brain. Substantial increases in malondialdehyde (by 59.6%–77.3%) and nitric oxide (by 43.7%–58.7%) were observed in the cortex, striatum, hippocampus, and medulla of rotenone-treated rats. Meanwhile, glutathione decreased by 24.3%–59.8% in these brain regions. Rotenone administration was associated with a significant decrease in paraoxonase 1 activity in the cerebral cortex, striatum, hippocampus, medulla, midbrain, and cerebellum (by 66%–73.6%). Further, acetylcholinesterase activity decreased by 44.4% in the cortex and Bcl-2 decreased in the striatum by 33.9% after rotenone injection. Rotenone induced a marked decrease in tyrosine hydroxylase immunoreactivity and increased both TNF-α and caspase-3 immunoreactivity in the striatum. These effects of rotenone were substantially reduced by coadministration of methylene blue, which resulted in significantly decreased malondialdehyde and nitrite and increased glutathione in different regions of the brain. In addition, there was increased paraoxonase 1 and acetylcholinesterase activity in response to treatment with methylene blue. Bcl-2 levels in the striatum increased significantly after the highest dose of methylene blue. Methylene blue also significantly decreased striatal expression of TNF-α and caspase-3 (apoptosis), and the degree of neuronal degeneration and gliosis in the striatum, substantia nigra, cortex, and hippocampus. Treatment with methylene blue resulted in an increased number of tyrosine hydroxylase-positive cells in the striatum, substantia nigra, and cerebral cortex, compared with the rotenone control group. The present findings suggest that treatment with methylene blue could prevent neuronal degeneration induced by rotenone injection in the rat brain. This effect may be mediated by inhibition of oxidative stress and apoptotic markers, and by enhancement of apoptotic markers and acetylcholinesterase activity.