Corrine Morrison, Dr. Scott Steffensen, Psychology and Neuroscience Center

Introduction:

An estimated seven to ten million people worldwide are living with Parkinson’s Disease (PD) and about 60,000 Americans are diagnosed with the disease each year (Parkinson’s Disease Foundation, 2013). The underlying cause of the movement disorders in PD is dopamine (DA) neuron degeneration in the substantia nigra pars compacta (SNc). A proposed mechanism behind DA cell death is additional oxidative stress that occurs in early stages of the disease state. NM is believed to be a protective agent against reactive oxygen species (ROS), but as NM depigmentation is a distinctive feature of PD, it is likely that there is a link between the loss of NM and increased oxidative stress. The goal of this study was to determine if NM could be induced in live brain tissue in an attempt to circumvent the natural depigmentation of the SNc in PD.

Background:

The decreased levels of NM in PD patients seem to be correlated with neuronal degeneration. A hallmark characteristic of this disease state is increased iron. In a healthy state, NM is considered an endogenous iron-binding molecule that may be the primary reservoir for iron in catecholaminergic cells to maintain homeostasis (Zecca et al., 2001). As the amount of NM present decreases, the existing NM will become saturated with iron leading to an increase of free iron, which is highly redox-reactive (Gerlach, Riederer, & Double, 2008). Unbound iron catalyzes the Fenton reaction, forming cytotoxic hydroxy radicals from hydrogen peroxide (Youdim, Ben-Shachar, & Riederer, 1989). This added oxidative stress could lead to DA cell death. Although, at physiological pH, iron is known to bind and complex with DA forming a possible intermediate leading to NM. NM is a radical scavenger that protects neurons against oxidative stress. We wanted to determine if we could induce NM in order to reverse the depigmentation of the SNc that happens during PD and protect the DA neurons. In preliminary in vitro studies, we found that iron markedly enhances melanin formation, which formed the rationale for pursing NM formation studies in brain slices.

Methodology:

Isoflurane-anesthetized mice were rapidly decapitated, and brain harvested. The mouse brain was dissected and rapidly cooled in a low Ca2+, high Mg2+ ice-cold solution. Slices 400 μm thick were then obtained using a vibratome sapphire blade. The slices were viewed under a microscope, while bathed in a continuous flow of artificial cerebrospinal fluid (ACSF) with either iron or oxyradical initiators while being held at a constant 35.7° C. We took time-lapse pictures of the slices every 2 minutes over a 5-hour period. Then we fixed the slices in formalin, saturate them in 30% sucrose, and cut them on cryostat (15 μm) at -20° C. Lastly, we stained the slices with the Fontana Masson stain as this is a silver stain that only stains melanins. The Fontana Masson stain uses ammoniacal silver solution, gold chloride, 2% aq. sodium thiosulphate, and a neutral red counter stain (Ellis, 2011). These slides were observed microscopically to confirm NM formation.

Results:

Unfortunately the results of the project were not as profound as we had hoped. The mouse brain slices were so thin that the process of the staining caused tearing and many of the slices became unusable for further observation. The slices that were salvageable proved to be difficult to analyze because the staining process made it hard to differentiate the brain anatomy. After looking through different brain atlases to aid in the search for the anatomy, we found one brain slice (Figure 1) that showed a small difference in darkness in the area we believe is the SNc, meaning NM formation did occur.

Discussion:

The brain slices did not show a significant difference in NM formation, which we believe was a result of insufficient time allowed for the NM to form. With this information in mind, further studies can be carried out in which thicker brain slices are taken to ensure the ability for complete staining without damaging the slices, then allotting more time for NM formation and a more extensive analysis of the NM positive cells in order to ensure that those formed are significantly different than control slices.

Conclusion:

Our results showed varying amounts of possible NM formation in the SNc. The preliminary research supported our methodology for inducing NM formation, and the results support the hypothesis that NM can be induced in live tissue. Although the results were not significant, further research is needed to understand the timing of the formation mechanism in order to produce more concrete results.

Sources:

Ellis, R. (2011). Masson Fontana Staining Protocol. Retrieved from http://www.ihcworld.com/_protocols/special_stains/masson_fontana_ellis.htm

Gerlach, M., Riederer, P., & Double, K. L. (2008). Neuromelanin-bound ferric iron as an experimental model of dopaminergic neurodegeneration in Parkinson’s disease. Parkinsonism Relat Disord, 14 Suppl 2, S185-188. doi: 10.1016/j.parkreldis.2008.04.028

Parkinson’s Disease Foundation, I. (2013). Statistics on Parkinson’s. Retrieved from http://www.pdf.org/en/parkinson_statistics

Youdim, M. B., Ben-Shachar, D., & Riederer, P. (1989). Is Parkinson’s disease a progressive siderosis of substantia nigra resulting in iron and melanin induced neurodegeneration? Acta Neurol Scand Suppl, 126, 47-54.

Zecca, L., Gallorini, M., Schunemann, V., Trautwein, A. X., Gerlach, M., Riederer, P., . . . Tampellini, D. (2001). Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes. J Neurochem, 76(6), 1766-1773.