中文摘要
性賀爾蒙已知在促進神經保護和神經再生中扮演重要的角色，但其作用機制和效果都還尚待釐清。在這項研究中，我們選擇斑馬魚作為主要模式生物，用來探討腦受創傷後複雜的修復過程。研究主要目的是分析性賀爾蒙如何調節成年斑馬魚於腦創傷後神經組織的修復與再生機制。在成年斑馬魚小腦由針穿刺產生外傷後，我們利用給予雌激素或以保康絲(柔沛; finasteride)抑制5α還原酶，調節實驗個體中性賀爾蒙的濃度與合成途徑。除了由組織切片染色追蹤修復過程，我們在創傷後的第3、6、和10天收取腦組織進行基因表現定量分析，藉以獲得目標基因的表現量變化數據。我們發現，經由保康絲(柔沛)的給予，神經再生的生物指標分子gfap和雌激素合成的關鍵酵素cyp19a1b mRNA表現量均被抑制。另一方面，雌激素的給予則會增加gfap和cyp19a1b mRNA的表現量。接著，我們分析了類固醇激素合成途徑的其它上游酵素的表達，包括cyp17和3β-hsd。出人意料的是，當給予保康絲(柔沛)後，斑馬魚小腦中cyp17和3β-hsd mRNA的表現量均出現顯著的增加，然而雌激素只會降低3β-hsd mRNA的表現量。這些結果意謂著雌激素可影響斑馬魚的神經再生且參與腦修復再生過程中的反饋調節機制。然而，如果抑制5α還原酶的活性，可能對腦的再生修復產生不利影響。類固醇合成酵素活性或表現量的調節，或有可能控制斑馬魚腦外傷後的神經修復再生。

Abstract
Sex hormones are known to play an important role in promoting neuroprotective activity and neurogenesis, but their roles and effects are not yet fully understood. Using zebrafish as a neurogenesis model organism, its brain serves as an excellent research system to study brain repair and the complex neurogenic process. In this study, we aimed to analyze how sex hormones regulate neurogenesis and brain regeneration after traumatic brain injury in adult zebrafish. After generating an injury by needle punch into the adult zebrafish cerebellum, we modified the sex hormonal level through estrogenic treatment or inhibition of 5α-reductase by finasteride treatment. At 3, 6 and 10 days post-injury, we harvested the fish brains for cell proliferation assay by histological analysis, and target gene expression profiling by qPCR assay. We found the mRNA expression level of neurogenesis biomarker gfap, and the key enzyme of estradiol synthesis cyp19a1b, were both down-regulated by finasteride. On the other hand, estradiol treatment increased mRNA expression of gfap and cyp19a1b. Next, we analyzed the expression of other upstream enzymes in the steroid hormone biosynthesis pathway, including cyp17 and 3β-hsd . Surprisingly, finasteride increased cyp17 and 3β-hsd mRNA expression, while estradiol treatment reduced 3β-hsd mRNA expression. The data suggests a feedback regulatory mechanism of estrogen during neurogenesis and brain regeneration. This study confirmed that estrogenic treatment can promote neurogenesis after traumatic brain injury in adult zebrafish. However, inhibition of 5α-reductase activity by finasteride may pose detrimental effect on brain regeneration. Altogether, we concluded that the regulation of steroidogenic enzymes may have a neurogenic switch mechanism on neurogenesis and after traumatic brain injury in zebrafish.

5. Reference
1 Xydakis, M. S., Ling, G. S. F., Mulligan, L. P., Olsen, C. H. & Dorlac, W. C. Epidemiologic aspects of traumatic brain injury in acute combat casualties at a major military medical center: A cohort study. Annals of Neurology 72, 673-681, doi:10.1002/ana.23757 (2012).
2 Faul, M., Xu, L., Wald, M. M., Coronado, V. & Dellinger, A. M. Traumatic Brain Injury in the United States: National Estimates of Prevalence and Incidence, 2002-2006. Injury Prevention 16, A268-A268, doi:10.1136/ip.2010.029951 (2010).
3 Lingsma, H. F. et al. Large between-center differences in outcome after moderate and severe traumatic brain injury in the international mission on prognosis and clinical trial design in traumatic brain injury (IMPACT) study. Neurosurgery 68, 601-607; discussion 607-608, doi:10.1227/NEU.0b013e318209333b (2011).
4 Margulies, S., Hicks, R. & Combination Therapies for raumatic Brain Injury Workshop, L. Combination therapies for traumatic brain injury: prospective considerations. J Neurotrauma 26, 925-939, doi:10.1089/neu.2008-079410.1089/neu.2008.0794 (2009).
5 Moretti, L. et al. Cognitive decline in older adults with a history of traumatic brain injury. Lancet Neurol 11, 1103-1112, doi:10.1016/S1474-4422(12)70226-0 (2012).
6 Greve, M. W. & Zink, B. J. Pathophysiology of Traumatic Brain Injury. Mount Sinai Journal of Medicine 76, 97-104, doi:10.1002/msj.20104 (2009).
7 Saatman, K. E. et al. Classification of traumatic brain injury for targeted therapies. Journal of Neurotrauma 25, 719-738, doi:10.1089/neu.2008.0586 (2008).
8 Jain, K. K. Neuroprotection in traumatic brain injury. Drug Discov Today 13, 1082-1089, doi:10.1016/j.drudis.2008.09.006 (2008).
9 Ginsberg, M. D. Current status of neuroprotection for cerebral ischemia: synoptic overview. Stroke 40, S111-114, doi:10.1161/STROKEAHA.108.528877 (2009).
10 Tolias, C. M. & Bullock, M. R. Critical appraisal of neuroprotection trials in head injury: what have we learned? NeuroRx 1, 71-79, doi:10.1602/neurorx.1.1.71 (2004).
11 Loane, D. J. & Faden, A. I. Neuroprotection for traumatic brain injury: translational challenges and emerging therapeutic strategies. Trends Pharmacol Sci 31, 596-604, doi:10.1016/j.tips.2010.09.005 (2010).
12 Skolnick, B. E. et al. A clinical trial of progesterone for severe traumatic brain injury. N Engl J Med 371, 2467-2476, doi:10.1056/NEJMoa1411090 (2014).
13 Xiong, Y., Mahmood, A. & Chopp, M. Angiogenesis, neurogenesis and brain recovery of function following injury. Curr Opin Investig Drugs 11, 298-308 (2010).
14 Lamprecht, M. R. & Morrison, B. A Combination Therapy of 17 beta-Estradiol and Memantine Is More Neuroprotective Than Monotherapies in an Organotypic Brain Slice Culture Model of Traumatic Brain Injury. Journal of Neurotrauma 32, 1361-1368, doi:10.1089/neu.2015.3912 (2015).
15 Bazer, F. W. et al. Novel pathways for implantation and establishment and maintenance of pregnancy in mammals. Mol Hum Reprod 16, 135-152, doi:10.1093/molehr/gap095 (2010).
16 Karasu, T., Marczylo, T. H., Maccarrone, M. & Konje, J. C. The role of sex steroid hormones, cytokines and the endocannabinoid system in female fertility. Hum Reprod Update 17, 347-361, doi:10.1093/humupd/dmq058 (2011).
17 Lephart, E. D. A review of brain aromatase cytochrome P450. Brain Res Rev 22, 1-26 (1996).
18 Brock, O., Keller, M., Veyrac, A., Douhard, Q. & Bakker, J. Short term treatment with estradiol decreases the rate of newly generated cells in the subventricular zone and main olfactory bulb of adult female mice. Neuroscience 166, 368-376, doi:10.1016/j.neuroscience.2009.12.050 (2010).
19 Bowers, J. M., Waddell, J. & McCarthy, M. M. A developmental sex difference in hippocampal neurogenesis is mediated by endogenous oestradiol. Biol Sex Differ 1, 8, doi:10.1186/2042-6410-1-8 (2010).
20 Mazzucco, C. A. et al. Both estrogen receptor alpha and estrogen receptor beta agonists enhance cell proliferation in the dentate gyrus of adult female rats. Neuroscience 141, 1793-1800, doi:10.1016/j.neuroscience.2006.05.032 (2006).
21 Ormerod, B. K. & Galea, L. A. Reproductive status influences cell proliferation and cell survival in the dentate gyrus of adult female meadow voles: a possible regulatory role for estradiol. Neuroscience 102, 369-379 (2001).
22 Fan, X., Warner, M. & Gustafsson, J. A. Estrogen receptor beta expression in the embryonic brain regulates development of calretinin-immunoreactive GABAergic interneurons. Proc Natl Acad Sci U S A 103, 19338-19343, doi:10.1073/pnas.0609663103 (2006).
23 Wang, L., Andersson, S., Warner, M. & Gustafsson, J. A. Estrogen receptor (ER)beta knockout mice reveal a role for ERbeta in migration of cortical neurons in the developing brain. Proc Natl Acad Sci U S A 100, 703-708, doi:10.1073/pnas.242735799 (2003).
24 Brinton, R. D. Estrogen-induced plasticity from cells to circuits: predictions for cognitive function. Trends Pharmacol Sci 30, 212-222, doi:10.1016/j.tips.2008.12.006 (2009).
25 Azcoitia, I., Arevalo, M. A., De Nicola, A. F. & Garcia-Segura, L. M. Neuroprotective actions of estradiol revisited. Trends in Endocrinology and Metabolism 22, 467-473, doi:10.1016/j.tem.2011.08.002 (2011).
26 Agis-Balboa, R. C. et al. Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 103, 14602-14607, doi:10.1073/pnas.0606544103 (2006).
27 Li, X., Chen, C. L., Singh, S. M. & Labire, F. The Enzyme and Inhibitors of 4-Ene-3-Oxosteroid 5-Alpha-Oxidoreductase. Steroids 60, 430-441, doi:Doi 10.1016/0039-128x(95)00021-H (1995).
28 Siiteri, P. K. & Wilson, J. D. Dihydrotestosterone in prostatic hypertrophy. I. The formation and content of dihydrotestosterone in the hypertrophic prostate of man. J Clin Invest 49, 1737-1745, doi:10.1172/JCI106391 (1970).
29 Geller, J., Albert, J., Lopez, D., Geller, S. & Niwayama, G. Comparison of Androgen Metabolites in Benign Prostatic Hypertrophy (Bph) and Normal Prostate. Journal of Clinical Endocrinology & Metabolism 43, 686-688 (1976).
30 Bartsch, G., Rittmaster, R. S. & Klocker, H. Dihydrotestosterone and the concept of 5alpha-reductase inhibition in human benign prostatic hyperplasia. World J Urol 19, 413-425 (2002).
31 Thompson, I. M. et al. The influence of finasteride on the development of prostate cancer. N Engl J Med 349, 215-224, doi:10.1056/NEJMoa030660 (2003).
32 Lephart, E. D. Age-related changes in brain and pituitary 5 alpha-reductase with finasteride (Proscar) treatment. Neurobiol Aging 16, 647-650 (1995).
33 Djebaili, M., Hoffman, S. W. & Stein, D. G. Allopregnanolone and progesterone decrease cell death and cognitive deficits after a contusion of the rat pre-frontal cortex. Neuroscience 123, 349-359 (2004).
34 Djebaili, M., Guo, Q. M., Pettus, E. H., Hoffman, S. W. & Stein, D. G. The neurosteroids progesterone and allopregnanolone reduce cell death, gliosis, and functional deficits after traumatic brain injury in rats. Journal of Neurotrauma 22, 106-118, doi:DOI 10.1089/neu.2005.22.106 (2005).
35 Brinton, R. D. & Wang, J. M. Preclinical analyses of the therapeutic potential of allopregnanolone to promote neurogenesis in vitro and in vivo in transgenic mouse model of Alzheimer's disease. Curr Alzheimer Res 3, 11-17 (2006).
36 Charalampopoulos, I. et al. Neurosteroids as endogenous inhibitors of neuronal cell apoptosis in aging. Neuroendocrine and Immune Crosstalk 1088, 139-152, doi:10.1196/annals.1366.003 (2006).
37 Duskova, M., Hill, M., Hanus, M., Matouskova, M. & Starka, L. Finasteride treatment and neuroactive steroid formation. Prague Med Rep 110, 222-230 (2009).
38 Marz, M., Schmidt, R., Rastegar, S. & Strahle, U. Regenerative response following stab injury in the adult zebrafish telencephalon. Dev Dyn 240, 2221-2231, doi:10.1002/dvdy.22710 (2011).
39 Kishimoto, N., Shimizu, K. & Sawamoto, K. Neuronal regeneration in a zebrafish model of adult brain injury. Dis Model Mech 5, 200-209, doi:10.1242/dmm.007336 (2012).
40 Sato, T., Takahoko, M. & Okamoto, H. HuC:Kaede, a useful tool to label neural morphologies in networks in vivo. Genesis 44, 136-142, doi:10.1002/gene.20196 (2006).
41 Kizil, C., Kaslin, J., Kroehne, V. & Brand, M. Adult neurogenesis and brain regeneration in zebrafish. Developmental Neurobiology 72, 429-461, doi:10.1002/dneu.20918 (2012).
42 Wu, C. C. et al. On the crucial cerebellar wound healing-related pathways and their cross-talks after traumatic brain injury in Danio rerio. PLoS One 9, e97902, doi:10.1371/journal.pone.0097902 (2014).
43 Zhao, Y. et al. Interactions between SIRT1 and MAPK/ERK regulate neuronal apoptosis induced by traumatic brain injury in vitro and in vivo. Exp Neurol 237, 489-498, doi:10.1016/j.expneurol.2012.07.004 (2012).
44 Kiryu-Seo, S. et al. Neuronal injury-inducible gene is synergistically regulated by ATF3, c-Jun, and STAT3 through the interaction with Sp1 in damaged neurons. J Biol Chem 283, 6988-6996, doi:10.1074/jbc.M707514200 (2008).
45 Barha, C. K. & Galea, L. A. Influence of different estrogens on neuroplasticity and cognition in the hippocampus. Biochim Biophys Acta 1800, 1056-1067, doi:10.1016/j.bbagen.2010.01.006 (2010).
46 Ormerod, B. K., Lee, T. T. & Galea, L. A. Estradiol initially enhances but subsequently suppresses (via adrenal steroids) granule cell proliferation in the dentate gyrus of adult female rats. J Neurobiol 55, 247-260, doi:10.1002/neu.10181 (2003).
47 Ormerod, B. K., Lee, T. T. & Galea, L. A. Estradiol enhances neurogenesis in the dentate gyri of adult male meadow voles by increasing the survival of young granule neurons. Neuroscience 128, 645-654, doi:10.1016/j.neuroscience.2004.06.039 (2004).
48 Le Page, Y., Vosges, M., Servili, A., Brion, F. & Kah, O. Neuroendocrine effects of endocrine disruptors in teleost fish. J Toxicol Environ Health B Crit Rev 14, 370-386, doi:10.1080/10937404.2011.578558 (2011).
49 Ge, S., Sailor, K. A., Ming, G. L. & Song, H. Synaptic integration and plasticity of new neurons in the adult hippocampus. J Physiol 586, 3759-3765, doi:10.1113/jphysiol.2008.155655 (2008).
50 Mirescu, C. & Gould, E. Stress and adult neurogenesis. Hippocampus 16, 233-238, doi:10.1002/hipo.20155 (2006).
51 Drake, L. et al. The effects of finasteride on scalp skin and serum androgen levels in men with androgenetic alopecia. J Am Acad Dermatol 41, 550-554 (1999).
52 Tobin, V. A. & Canny, B. J. Testosterone regulates gonadotropin-releasing hormone-induced calcium signals in male rat gonadotrophs. Endocrinology 137, 1299-1305, doi:10.1210/endo.137.4.8625903 (1996).
53 Sarachana, T. & Hu, V. W. Differential recruitment of coregulators to the RORA promoter adds another layer of complexity to gene (dys) regulation by sex hormones in autism. Mol Autism 4, 39, doi:10.1186/2040-2392-4-39 (2013).
54 Sarachana, T., Xu, M., Wu, R. C. & Hu, V. W. Sex hormones in autism: androgens and estrogens differentially and reciprocally regulate RORA, a novel candidate gene for autism. PLoS One 6, e17116, doi:10.1371/journal.pone.0017116 (2011).
55 Martino, G., Pluchino, S., Bonfanti, L. & Schwartz, M. Brain regeneration in physiology and pathology: the immune signature driving therapeutic plasticity of neural stem cells. Physiological reviews 91, 1281-1304, doi:10.1152/physrev.00032.2010 (2011).
56 Xiao, G., Wei, J., Yan, W., Wang, W. & Lu, Z. Improved outcomes from the administration of progesterone for patients with acute severe traumatic brain injury: a randomized controlled trial. Critical care 12, R61, doi:10.1186/cc6887 (2008).
57 Wright, D. W. et al. ProTECT: a randomized clinical trial of progesterone for acute traumatic brain injury. Annals of emergency medicine 49, 391-402, 402 e391-392, doi:10.1016/j.annemergmed.2006.07.932 (2007).
58 Voskuhl, R. R. et al. Estriol combined with glatiramer acetate for women with relapsing-remitting multiple sclerosis: a randomised, placebo-controlled, phase 2 trial. The Lancet. Neurology 15, 35-46, doi:10.1016/S1474-4422(15)00322-1 (2016).
59 Stein, D. G. Embracing failure: What the Phase III progesterone studies can teach about TBI clinical trials. Brain injury 29, 1259-1272 (2015).
60 Engler-Chiurazzi, E. B., Brown, C. M., Povroznik, J. M. & Simpkins, J. W. Estrogens as neuroprotectants: Estrogenic actions in the context of cognitive aging and brain injury. Progress in neurobiology, doi:10.1016/j.pneurobio.2015.12.008 (2016).
61 Diotel, N. et al. Activity and expression of steroidogenic enzymes in the brain of adult zebrafish. The European journal of neuroscience 34, 45-56, doi:10.1111/j.1460-9568.2011.07731.x (2011).