PROGESTERONE ATTENUATES DEMYELINATION AND MICROGLIAL REACTION IN THE LYSOLECITHIN-INJURED SPINAL CORD L. GARAY, a,b V. TÜNGLER, a,c,d M. C. G. DENISELLE, a,b A. LIMA, a P. ROIG a AND A. F. DE NICOLA a,b * a Laboratory of Neuroendocrine Biochemistry, Instituto de Biologia y Medicina Experimental-CONICET, Obligado 2490, 1428 Buenos Ai- res, Argentina b Department of Human Biochemistry, Faculty of Medicine, University of Buenos Aires, Paraguay 2155, 1425 Buenos Aires, Argentina c Institut für Pharmakologie und Toxikologie, Charité - Universitätsme- dizin Berlin, Dorotheenstr. 94, 10117 Berlin, Germany d Klinik für Kinder- und Jugendmedizin, Technische Universität Dres- den, Fetscherstr. 74, 01307, Dresden, Germany Abstract—Progesterone treatment of mice with experimental autoimmune encephalomyelitis has shown beneficial effects in the spinal cord according to enhanced clinical, myelin and neu- ronal-related parameters. In the present work, we report proges- terone effects in a model of primary demyelination induced by the intraspinal injection of lysophospatidylcholine (LPC). C57Bl6 adult male mice remained steroid-untreated or received a single 100 mg progesterone implant, which increased circu- lating steroid levels to those of mouse pregnancy. Seven days afterwards mice received a single injection of 1% LPC into the dorsal funiculus of the spinal cord. A week after, anesthetized mice were perfused and paraffin embedded sections of the spinal cord stained for total myelin using Luxol Fast Blue (LFB) histochemistry, for myelin basic protein (MBP) immunohisto- chemistry and for determination of OX-42 microglia/macro- phages. Cryostat sections were also prepared and stained for oligodendrocyte precursors (NG2 cells) and mature oligoden- drocytes (CC1 cells). A third batch of spinal cords was pre- pared for analysis of the microglial marker CD11b mRNA using qPCR. Results showed that progesterone pretreatment of LPC- injected mice decreased by 50% the area of demyelination, evaluated by either LFB staining or MBP immunostaining, in- creased the density of NG2 cells and of mature, CC1 oligo- dendrocytes and decreased the number of OX-42 cells, re- spect of steroid-untreated LPC mice. CD11b mRNA was hyper- expressed in LPC-treated mice, but significantly reduced in LPC-mice receiving progesterone. These results indicated that progesterone antagonized LPC injury, an effect involving (a) increased myelination; (b) stimulation of oligodendrocyte pre- cursors and mature oligodendrocytes, and (c) attenuation of the microglial/macrophage response. Thus, use of a focal demyeli- nation model suggests that progesterone exerts promyelinating and anti-inflammatory effects at the spinal cord level. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: demyelination, lysolecithin, microglial reaction, progesterone, remyelination. Multiple sclerosis (MS) is a major neurodegenerative disease that causes neurological disability in an estimated 2.5 million people worldwide. MS presents a gender preference, with a woman to man ratio of 2.6, although the incidence in women increases after menopause (Confavreux et al., 1998; Hughes, 2004). Interestingly, hormonal factors have been considered to play a pathogenic role in MS. Early studies report an association between lesion size of MS patients, low circulating progesterone levels and high estradiol levels dur- ing the sex cycle, suggesting a beneficial role of progester- one (Bansil et al., 1999; Pozzilli et al., 1999). The PRIMS (Pregnancy in Multiple Sclerosis) study concluded that re- lapses of MS are less frequent during the last trimester of pregnancy but reappear following delivery, supporting a pro- tective role of sex steroids (El-Etr et al., 2005). This evidence led to the suggestion that progesterone-induced immunosup- pression during human pregnancy may prevent relapses of MS (Confavreux et al., 1998; Hughes, 2004; Druckmann and Druckmann, 2005). Conclusive demonstrations regarding the neuroprotec- tive and promyelinating role of progesterone in the central and peripheral nervous system (Melcangi et al., 2000; Azcoi- tia et al., 2003; Brinton et al., 2008; Schumacher et al., 2008; De Nicola et al., 2009), stimulated trials on its therapeutic value for MS and models of the disease such as experimental autoimmune encephalomyelitis (EAE) and cuprizone-in- duced demyelination. Earlier reports have shown variable effects of progesterone in EAE, ranging from inactivity, in- creased vulnerability of neurons to disease improvement if estradiol is combined with progesterone (Kim et al., 1999; Bebo et al., 2001; Hoffman et al., 2001). In particular, the synthetic progestin medroxyprogesterone acetate has shown a protective effect in EAE (Elliot et al., 1973). More recent evidence supports that progesterone pro- vides beneficial effects to rodents with EAE. For example, progesterone treatment prior to EAE induction with myelin oligodendrocyte glycoprotein (MOG) attenuates the clinical scores of the disease, slightly delays disease onset and decreases demyelination foci, according to Luxol Fast Blue staining (LFB), myelin basic protein (MBP) and proteolipid protein (PLP) protein and mRNA expression. Key genes of motoneuron function and axonal parameters are also en- hanced in EAE mice receiving progesterone (Garay et al., 2007, 2008, 2009). Another group has shown that progester- one given at the time of EAE induction reduces peak score and the cumulative disease index, decreases proinflamma- tory and increases antiflammatory chemokine secretion *Correspondence to: A. F. De Nicola, Laboratory of Neuroendocrine Biochemistry, Instituto de Biologia y Medicina Experimental, Obligado 2490, 1428 Buenos Aires, Argentina. Tel: +54-11-47832869; fax: +54-11-47862564. E-mail address: alejandrodenicola@gmail.com (A.F. De Nicola). Abbreviations: EAE, experimental autoimmune encephalomyelitis; LFB, Luxol Fast Blue; LPC, lysophospatidylcholine; MBP, myelin basic protein; MS, multiple sclerosis; OPC, oligodendrocyte precursor cells; PBS, phosphate-buffered saline; PFA, paraformaldehyde. Neuroscience 192 (2011) 588 –597 0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.06.065 588