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.

We investigated the effect of cerebrolysin, a peptide mixture used for promoting memory and recovery from cerebral stroke, on the development of oxidative stress and nigrostriatal cell injury induced by rotenone administration in rats. Rotenone 1.5 mg/kg was given subcutaneously three times weekly either alone or in combination with cerebrolysin at 21.5, 43, or 86 mg/kg. Rats were euthanized 14 days after starting the rotenone injection. Lipid peroxidation (malondialdehyde), reduced glutathione (GSH), nitric oxide (nitrite) concentrations, paraoxonase 1 (PON1), and acetylcholinesterase (AChE) activities – as well as the monocyte chemoattractant protein-1 (MCP-1) and the antiapoptotic protein Bcl-2 – were measured in the brain. Histopathology, tyrosine hydroxylase, inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and cleaved caspase-3 immunohistochemistry were also performed. Rotenone caused a significantly elevated oxidative stress and proinflammatory response in the different brain regions. Malondialdehyde and nitric oxide concentrations were significantly increased, while GSH markedly decreased in the cerebral cortex, striatum, hippocampus, and in the rest of the brain. PON1 and AChE activities significantly decreased with respect to the control levels after rotenone application. Striatal Bcl-2 was significantly decreased while MCP-1 increased following rotenone injection. Rotenone caused prominent iNOS, TNF-α, and caspase-3 immunostaining in the striatum and resulted in markedly decreased tyrosine hydroxylase immunoreactivity in the substantia nigra and striatum. Cerebrolysin coadministered with rotenone decreased lipid peroxidation, increased GSH, and inhibited the elevation of nitric oxide induced by rotenone. Cerebrolysin also decreased the rotenone-induced decline in the PON1 and AChE activities and the rotenone-mediated changes in the striatal Bcl-2 and MCP-1 levels. The drug reduced iNOs, TNF-α, and caspase 3 expressions and increased the tyrosine hydroxylase immunoreactivity in the striatum. Cerebrolysin markedly prevented the development of neuronal damage in the cortex and striatum. These data suggest that cerebrolysin may have potential therapeutic effect in Parkinson’s disease.