Bacterial melanin in rat models of Parkinson's disease: a potential neuroprotective strategy

Melanins are widely used in medicine, pharmacology and cosmetics. Different technologies have been used to obtain melanin including: chemical synthesis based on oxidation of tyrosine and its derivatives; extraction from animal materials; alkaline extraction from plant material; and microbiological synthesis. A few number of works have been published that were focused on purification of water insoluble 3,4-dihydroxy-phenylalanine-melanins (Kukulianskaia et al., 2002). The majority of synthetic and natural melanins are insoluble in water that significantly complicates preparation of pharmacological and cosmetic preparations. Obtaining of low-cost soluble biotechnological melanin can speed up application of melanin in medicine and other fields. For the first time, melanin-synthesizing strain with high level of pigment synthesis – Bacillus thuringiensis was obtained. The ecologically safe technology of biosynthesis, isolation and purification of the bacterial melanin has been elaborated. Melanin metabolism disorders can be involved in the etiology of such diseases as parkinsonism, senile macular degeneration, and senile deafness. This pigment is also relevant to the wellknown association between pigmentary abnormalities and deafness (Warrensburg’s and Usher’s syndromes). The Alzheimer’s disease and Down syndrome were observed to be also accompanied with pathological disorders in melanin metabolism. Parkinson’s disease (PD) is caused by a deficiency of the neurotransmitter dopamine at the nerve terminals of the nigrostriatal dopaminergic neurons in the striatum, due to selective loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc). Majority of PD cases are sporadic and age related, and approximately 5% are classified as familial disease. Although subject to intensive research, the etiology of PD is not completely studied and the treatment is basically symptomatic. Many factors are speculated to operate in the mechanism of cell death of the nigrostriatal dopaminergic neurons in PD, including oxidative stress and cytotoxicity of reactive oxygen spices, disturbances of intracellular calcium homeostasis, exogenous and endogenous toxins, and mitochondrial dysfunction. Neurodegenerative processes are generally characterized by a long-lasting course of neuronal death. Evidence of apoptotic cell death in various neuronal and non-neuronal cells was seen through DNA fragmentation and typical morphological changes in PD brains. Although the concept of programmed cell death (apoptosis) in PD is still controversial, data from postmortem brains of PD patients, animal models, and in vitro culture studies indicate the presence of apoptotic cell death as well as a proapoptotic environment in the nigrostriatal region in PD (Lev et al., 2003). Different concentrations of bacterial melanin have been tested in our experiments. It was revealed that the recovery period of motor functions in rats, dosed with various concentrations of bacterial melanin, was different in rats injected with bacterial melanin after unilateral ablation of sensorimotor cortex. In general the recovery of initially elaborated balancing motor reflex (Gevorkyan et al., 2007) occurred earlier in rats injected with low concentrations of bacterial melanin. When injected with low concentrations of melanin (6, 4.5 mg/mL) the recovery periods for instrumental conditioned reflex and balancing limb movements were significantly shorter. Studies have also shown that neuromelanin containing dopaminergic neurons of the SNc are more subjected to degeneration in patients with PD than dopaminergic neurons that do not contain melanin. The authors have shown that the free extracellular neuromelanin and microgliosis are the main causes of PD. The latest data show that the human extracellular neuromelanin in the absence of microglia itself is not toxic for neurons. But release of neuromelanin from destructed neurons causes the activation of microglia and subsequent neurodegeneration, proving that melanin containing neurons of substantia nigra (SN) are targeted in PD. Bacterial melanin has proved to be non toxic, and it does not cause activation of microglia when applied directly to brain tissue or after injections. It is proved that inflammatory factors may lead to the death of DA neurons in SNc. Bacterial melanin supports the survival of neurons in SNc after induced destruction and preserves dopaminergic cell bodies. Bacterial melanin causes dilation of capillaries in the lesion area, which increases the blood flow in the brain tissue (Petrosyan et al., 2012). Numbers of studies have shown neuroprotective action of melanocyte-stimulating-hormone on motor recovery following central nervous system lesion (Chen et al., 2008). There are several animal models of PD, such as PD mice produced by repeated injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; hemiparkinsonian rats produced by injecting 6-hydroxydopamine into one side of the ventrotegmental bundle; and electrolytic destruction of dopaminergic neurons in SN. In all experimental series of our project (destruction of cerebellar nuclei, SNc, etc.) we have used electrolytic destruction. The same method was also used for the present study. Excitotoxic lesions potentially have advantages over the applied electrolytic destruction, except for the possible chemical interaction and neuropeptide changes. Potential neuroprotective action of bacterial melanin was studied in rats after unilateral destruction of SNc dopaminergic neurons. Rats were initially trained to an instrumental conditioned reflex and then were subjected to unilateral electrolytic destruction of SNc. Unilateral deficit in balancing hindlimb movements was observed in all rats after the destruction. On the next day after the destruction, part of the animals (experimental group) was intramuscularly injected with bacterial melanin solution at the concentration 6 mg/mL (0.17 g/kg). The other group of operated rats served as a control. On the second day after the operation the testing of instrumental conditioned reflex was resumed in both groups. Comparison of recovery