1. UVR Exposure Affects Demyelination Rates in a Multiple Sclerosis Model Robert Hand Department of Biological Sciences, York College of Pennsylvania Project Summary Multiple sclerosis is regarded as an autoimmune disease due to the breakdown of protective myelin sheaths of CNS axons. Research suggests that, although the cause of MS is unknown, there may be benefits in having increased vitamin D levels through diet and ultraviolet radiation. In the proposed experiment, 49 Hr Hr (hairless) mice will have mild experimental autoimmune encephalomyelitis induced by injection. EAE will serve to model multiple sclerosis and its associated demyelination. Over the course of 180 days the mice will be exposed to constant UVR lighting in varying doses ranging from 0 hours (total blackness) to 24 hours in 4hr intervals. Blood will be drawn biweekly and levels of vitamin D 3 metabolite will be determined by liquid chromatography-tandem mass spectrometry. A terminal lumbar puncture will sample cerebrospinal fluid for the detection of myelin basic protein, which indicates the breakdown of myelin, and levels will be determined by a sandwich ELISA assay. Lastly, specimen spinal cords will be fixed and stained for histopathological analysis. Increasing UVR exposure rates are expected to yield increased vitamin D 3 metabolite levels and decreased MBP levels. Introduction Multiple sclerosis (MS) is a debilitating, demyelinating disease of the central nervous system. The cause of onset is unknown but it is understood that multiple sclerosis causes lesions to form in the protective myelin sheath of Schwann cells. This leads to decreased neural function. MS is often observed in fair-skinned individuals of northern European origin (Nosesworthy 1999). While a direct connection has not been made, the effect of melanin on sunlight absorption and vitamin D production is thought to be the reason for this disparity (Dumas and Jauberteau-Marchan 2000). From previous studies’ data regarding vitamin D, multiple sclerosis, and demyelination rates, one hypothesis is that increased UVR exposure will result in decreased demyelination rates. Direct injections of vitamin D 3 metabolite have been shown to reduce demyelination, and even increase remyelination, in an experimental acquired encephalomyelitis (EAE) mouse model (Nashold et al. 2000); therefore, naturally acquired vitamin D will theoretically reduce demyelination in this same multiple sclerosis model. Blood levels of vitamin D 3 metabolite, cerebrospinal fluid levels of myelin basic protein, and a post-mortem histopathological analysis of spinal cord tissue will be taken from each specimen in order to determine the affects of the various UVR exposure times on demyelination rate. The goal of this study is to provide a baseline for further studies in regards to prevention, treatment, and possible alleviation of multiple sclerosis through natural, environmental vitamin D supplementation. Review of Literature Multiple Sclerosis -The exact pathogenesis of multiple sclerosis is unknown. It is understood that genetic and environmental factors both contribute to multiple sclerosis (Ebers 2008) -Multiple sclerosis is more common in temperate climates, with an observed prevalence of 1/1000, than in tropical climates, where prevalence exists as 1/10000 (Ebers 2008) -An increased risk of MS was observed with shared genetic information within a family. Risks of dizygotic twins and siblings was determined to be smaller, but still 20-40x greater than that of the general population (Ebers et al. 1986) -Myelin Basic Protein (MBP) levels greater than 9ng/mL indicate active demyelination (Ohta et al. 2000) UVR Exposure and Vitamin D -UV radiation of the skin is the major source of vitamin D in animals (Holick 1995) -The most accurate indicator of vitamin D status is serum 25-hydroxyvitamin D (Snellman et al. 2009) -The best concentrations of serum (in humans) are between 90-100 nmol/L (Bischoff-Ferrari et al. 2006) -After reviewing over 7 million US military personnel (with at least 1 serum sample stored in the DoDSR), it its hypothesized that the risk of MS decreased due to increasing serum levels of 1,25-hydroxyvitamin D (Munger et al. 2006) -It is hypothesized that sunlight may be protective in MS due to the immunoregulatory functions of 1,25-dihydroxyvitamin D 3 observed in EAE mice (Nashold et al. 2000) Research Design Objectives -To determine a baseline exposure time required to achieve sufficient vitamin D metabolite levels (90-100 nmol/L) -To determine if naturally synthesized vitamin D (from sunlight exposure) is effective as a long-term defense against demyelination Hypothesis H R : There is a significant difference in the rate of demyelination, due to the resultant levels of vitamin D, from varying UVR exposure lengths Hypothetical Results Conclusion Sufficient dietary vitamin D was obtained by all mice due to the standardized diet; however, the various UVR exposure times caused for variation in overall vitamin D levels. Mice with higher levels of vitamin D, directly due to increased UVR exposure, exhibited significantly less demyelination. Thus, daily UVR exposure greater than 8hrs is necessary to generate demyelination-preventative levels of vitamin D Future Studies If this proposal materializes, it would help model data that allows for the calculation of human baselines. This would allow for crucial human trial studies. Literature Cited Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. 2006. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. American Journal of Clinical Nutrition 84:18- 28 Dumas, M., and M. O. Jauberteau-Marchan. "The Protective Role of Langerhans' Cells and Sunlight in Multiple Sclerosis." Medical Hypotheses 55.6 (2000): 517-20. PubMED. Web. 22 Jan. 2009. Ebers, George. 2008. Environmental factors and multiple sclerosis. Lancet Neurology 7:268-277 Ebers, G., Bulman, D., Sadovnick, A., Paty, D., Warren, S., Hader, W., Murray, T., Seland, T., Duquette, P., Grey, T., Nelson, R., Nicolle, M. and Brunet, D. 1986. A population-based study of multiple sclerosis. New England Journal of Medicine 6:1638-1642 Holick, M. F. 1995. Vitamin D and bone health. Symposium: “Nutritional Advances in Human Bone Metabolism”, American Institute of Nutrition. Experimental Biology '95 annual meeting, Atlanta, GA: 11595–11645 Munger, K., Levin, L., Hollis, B., Howard, N. and Ascherio, A. 2006. Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. The Journal of the American Medical Association 296(53):2832-2838 Nashold, F., Miller, D. and Hayes, C. 2000. 1,25-Dihydroxyvitamin D3 treatment decreases macrophage accumulation in the CNS of mice with experimental autoimmune encephalomyelitis. Journal of Neuroimmunology 103:171-179 Noseworthy, John H. 1999. Progress in determining the causes and treatment of multiple sclerosis. Nature 399:40- 47 Ohta, M., Ohta, K., Ma, J., Takeuchi, J., Saida, T., Nishimura, M. and Itoh, N. 2000. Clinical and analytical evaluation of an enzyme immunoassay for myelin basic protein in cerebrospinal fluid. Clinical Chemistry 46:1326- 1330 Acknowledgements I would like to thank Dr. Ronald Kaltreider for his continued support and mentoring of this project. I would also like to thank Dr. Bradley Rehnberg for helping construct this proposal in ISR of Fall 2008. 7896 Terminal Lumbar Puncture ~Day 180 (6 mo.) MBP analysis via ELISA Histopathological Analysis Spinal Cord (t.s.) Lieb Erichrome - MBP 49 Hr Hr Mice of Same Age Group 4: 7 Mice 12hr UVR Group 3: 7 Mice 8hr UVR Group 2: 7 Mice 4hr UVR Group 1: 7 Mice 0hr UVR Group 5: 7 Mice 16hr UVR Group 6: 7 Mice 20hr UVR Group 7: 7 Mice 24hr UVR EAE Induction Blood Collection from Tail Every 14 days 25OHD analysis via LC-MS http:// www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=stryer&part=A3653&rendertype=figure&id=A3670