Neuroprotective effect of metformin in MPTP-induced Parkinson’s disease in mice
Introduction
Currently metformin is gaining the attraction of researchers because of its incredible therapeutic values. At present metformin is globally one of the most generally recommended medications in the treatment of Diabetes mellitus (Chakraborty et al., 2011a, Chakraborty et al., 2011b). Metformin ameliorates high blood sugar without stimulating insulin secretion or causing hypoglycemia. Its long history of efficacy and safety has made this small drug molecule the most commonly prescribed medication. In addition to its antidiabetic potential, metformin has been proved to be a therapeutically effective drug candidate in various CNS disorders. (Li et al., 2010, Ng et al., 2012, Ashabi et al., 2014). Metformin, a potent AMPK activator was found to alleviate dopaminergic dysfunction and mitochondrial abnormalities in drosophila models of Parkinson’s disease (PD) (Ng et al. (2012)). Metformin is found to be neuroprotective by inhibiting apoptosis in neuronal cortical cells (El-Mir et al., 2008). Clinical trials conducted in Taiwanese population showed that metformin reduces the risk of PD occurring with type-II diabetes (Wahlqvist et al., 2011). Recently it was proposed that metformin promotes neurogenesis and enhances the spatial memory formation (Wang et al. (2012)). It was also observed that long-term treatment with metformin improves the healthspan and lifespan in mice (Martin-Montalvo et al., 2013). Previous studies suggest that metformin prevents the oxidative stress-related cellular death in non-neuronal cell lines (Labuzek et al., 2010a, Labuzek et al., 2010b). In addition to this metformin has inhibitory role on inflammatory transcription factors like nuclear factor-κB (Hirsch et al., 2013). Moreover, study conducted by Labuzek et al., 2010a, Labuzek et al., 2010b evinced that orally administered metformin rapidly crosses the blood–brain barrier and is distributed to various regions in the brain. Considering these facts, the primary objective of the present study was to evaluate the neuroprotective activity of metformin with respect to PD.
PD is a common, slowly progressive, neurodegenerative disease resulting from the degeneration of dopaminergic neurons in the substantia nigra (SN), a region of the brain that controls movement (Meissner et al., 2011). It is an age-related disorder characterized by tremors, muscular rigidity, bradykinesia and postural abnormalities (Meissner et al., 2011). Even though etiology of the disease remains unknown, there are many potential causes including endogenous and genetic factors or environmentally derived neurotoxins. Although the symptoms can be treated using currently available dopamine replacement strategies the present therapy to treat PD is not effective due to prolonged progress of the disease over several years. Moreover, chronic treatment with Dopamine agonist or l-dihydroxyphenylalanine (l-DOPA) results in severe motor and non-motor adverse effects (Meissner et al., 2011). Hence the drug with benefits of having neuroprotective and/or disease-modifying effects will be a major breakthrough in the PD treatment.
In this study, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, Sigma-Aldrich, St. Louis, MO, USA) which was used to induce dopaminergic toxicity is known to produce neuropathologic abnormalities resembling the idiopathic PD in humans (Javitch et al., 1984). To determine behavioral pattern, motor co-ordination tests like rotarod and open-field were carried out. Dopaminergic neurodegeneration was evaluated by using nissl staining and tyrosine hydroxylase (TH)-positive cells in SN-pars compacta (SNpc) using immunohistochemistry. Furthermore, the oxidative stress level in the midbrain was investigated by measuring the activities of superoxide dismutase (SOD), catalase (CAT) and levels of glutathione (GSH), lipid peroxidation (LPO). In addition the neurotrophic activity was assessed by determining the brain-derived neurotrophic factor (BDNF) levels.
Section snippets
Experimental procedures
Metformin was obtained as a gift sample from Lupin Pharmaceuticals, Inc. India. MPTP hydrochloride (MPTP-HCl) was purchased from Sigma–Aldrich (USA). All other chemicals and reagents used in the experiments were of analytical grade. Primary monoclonal mouse antibodies of TH and BDNF were used for the immunohistochemistry analysis.
Rotarod test
Fig. 1 depicts the results obtained by performing rotarod test. There was a significant motor impairment on day 4 as well as on day 20 in MPTP group when compared to normal control (P < 0.001). Metformin significantly improved the rotarod performance of the animals on day 20 (P < 0.01) while no significant improvement was observed on day 4.
Open-field test
Open-field test includes various parameters like line crossings, rearing, grooming and immobility. There was a significant decrease in the locomotor activity,
Discussion
Results of the present study provide strong evidence of a direct neuroprotective effect of metformin. This study illustrates the enhanced muscular and locomotor activity in MPTP-induced parkinsonian mice after a long-term metformin treatment. Furthermore its neuroprotective role was supported by reduced brain oxidative stress and preserved TH-positive dopaminergic neurons. The study gives an overview of neurotrophic potential of metformin which is manifested by significant increase in BDNF
Conclusion
We found that because of metformin treatment recovery occurs in parkinsonian mice, and is associated with benefits, including reduced oxidative stress and increased antioxidant defenses, leading to lower oxidative damage accumulation. In addition to neuroprotective activity it may possess neurotrophic potential. This study provides a strong preclinical support for the therapeutic potential of this compound in the treatment of PD.
Acknowledgements
This work was supported by the Department of Science and Technology (DST), New Delhi, India. Authors would like to thank Mr. Jayant Sancheti, Mr. Rahul Chaudhari, Mr. Gauresh Somani, Mr. Sagar Bacchav, Ms. Divya Kanchan, Ms. Vaibhavi Peshattiwar, Mr. Devang Sarvaiya and Mr. Madhav Seervi for their assistance in the experiment.
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