The Effect of 17β- and 17α-Estradiol on Myelination and Remyelination in Cerebellar Slices by Cassandra Joy Belo A thesis submitted in conformity with the requirements for the degree of Master of Science Laboratory Medicine and Pathobiology University of Toronto © Copyright by Cassandra Joy Belo 2013 The Effect of 17β- and 17α-Estradiol on Myelination and Remyelination in Cerebellar Slices Cassandra Joy Belo Master of Science Laboratory Medicine and Pathobiology University of Toronto 2013 Abstract Multiple sclerosis is thought to be an autoimmune disease that causes lesions and demyelination of axons in the central nervous system. A reduction in relapses is seen in the third trimester of pregnancy when estrogen levels are highest, followed by an increase in relapses in the first three months post-partum when estrogen levels drop. This thesis focuses on the effect of 17beta- and 17alpha-estradiol on myelination and remyelination in cultured rat cerebellar slices. No reproducible effect of 17beta-estradiol on myelination was detected. However, during lysolecithin-induced demyelination it had a possible protective effect in males and females, although it was not statistically significant. During myelination, 100 nM 17alpha- estradiol caused a small but significant decrease in MBP expression in females. During lysolecithin- induced demyelination, it had a possible, but not statistically significant, protective effect in males. Neither 17beta- nor 17alpha-estradiol had a reproducible or significant effect on remyelination in males or females. ii Acknowledgments I would firstly like to thank Dr. Joan Boggs for taking me on as her final graduate student. Your guidance has made me a better writer and scientist. I would also like to thank the members of the Boggs’ lab, Yuanfang Liu, Godha Rangaraj and Lopamudra Homchaudhuri who have helped me by answering my questions, talking through experiments, listening to all of my presentations and being excellent colleagues to enjoy lunch with. I would also like to thank my friends and family for supporting me through my research. My grandparents for their constant interest in how my research was going and for giving me practice talking about my work. Thank you to Chris for being so understanding and letting me put my research first and knowing when I needed a break. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii Abbreviations ................................................................................................................................. ix Chapter 1 Introduction .................................................................................................................... 1 1.1 Multiple Sclerosis ............................................................................................................... 1 1.2 Hormones and MS .............................................................................................................. 3 1.3 Hormone Concentrations .................................................................................................... 6 1.4 Hormone synthesis ............................................................................................................ 10 1.5 Estrogen and Progesterone Receptors ............................................................................... 12 1.6 Myelination and Remyelination ........................................................................................ 14 1.7 Effects of Estrogens .......................................................................................................... 18 1.7.1 Immune System .................................................................................................... 18 1.7.2 CNS Cultured Cells ............................................................................................... 19 1.7.3 CNS Co-cultures and Brain Slices ........................................................................ 22 1.7.4 Animal Trials ........................................................................................................ 23 1.7.5 Clinical Trials ........................................................................................................ 26 1.8 Hypothesis ......................................................................................................................... 27 1.9 Experimental Approach .................................................................................................... 27 Chapter 2 Materials and Methods ................................................................................................. 32 2.1 Slice culture ...................................................................................................................... 32 2.2 Remyelination in slices ..................................................................................................... 32 2.3 Chemicals and Antibodies ................................................................................................ 33 iv 2.4 Western Blotting ............................................................................................................... 33 2.5 Immunohistochemistry ..................................................................................................... 34 2.6 Image Analysis .................................................................................................................. 35 2.7 Statistical Analysis ............................................................................................................ 35 Chapter 3 Results .......................................................................................................................... 36 3.1 Optimization of brain slice culture conditions .................................................................. 36 3.2 Effect of estrogens and progesterone on myelination ....................................................... 38 3.2.1 Effect of 17β-estradiol on myelination in cerebellar brain slices ......................... 39 3.2.2 Effect of 17β-estradiol and progesterone in cerebellar brain slices ...................... 47 3.2.3 Effect of 17α-estradiol on myelination in cerebellar brain slices ......................... 49 3.3 Effect of estrogens on remyelination after lysolecithin demyelination ............................ 53 3.3.1 Optimization of demyelination and remyelination conditions .............................. 53 3.3.2 Effect of 17β-estradiol after lysolecithin treatment on remyelination .................. 56 3.3.3 Effect of 17α-estradiol after lysolecithin treatment on remyelination .................. 59 Chapter 4 Discussion .................................................................................................................... 63 4.1 Quantifying myelination and remyelination ..................................................................... 65 4.2 Effect of estradiol and progesterone on myelination in cerebellar brain slices ................ 65 4.2.1 Effect of 17β-estradiol on myelination ................................................................. 65 4.2.2 Effect of 17α-estradiol on myelination ................................................................. 66 4.2.3 Effect of combined treatment with 17β-estradiol and progesterone on myelination ........................................................................................................... 67 4.3 Effect of estradiol treatment on remyelination in cerebellar brain slices ......................... 69 4.3.1 Effect of 17β-estradiol on demyelination and remyelination ............................... 70 4.3.2 Effect of 17α-estradiol on demyelination and remyelination ............................... 72 4.3.3 Immunohistochemistry analysis of demyelination and remyelination ................. 72 4.4 Myelination versus remyelination ..................................................................................... 74 v 4.5 Influence of ethanol .......................................................................................................... 74 4.6 Advantages and disadvantages of cerebellar brain slices ................................................. 76 4.7 Variations in trials and western blotting ........................................................................... 77 4.8 Variations in immunohistochemistry ................................................................................ 79 4.9 Conclusions ....................................................................................................................... 80 4.10 Future directions ............................................................................................................... 81 References ..................................................................................................................................... 83 vi List of Tables Table 1………………………………………………………………………………………..…...8 Table 2…………………………………………………………………………………………...33 vii List of Figures Figure 1: Four clinical subtypes