Research ReportProtective effects of progesterone administration on axonal pathology in mice with experimental autoimmune encephalomyelitis
Introduction
Experimental autoimmune encephalomyelitis (EAE) is an immune-mediated disease that exhibits characteristics in common with Multiple Sclerosis (MS) (Ayers et al., 2004; Kornek et al., 2006; Wujek et al., 2002). EAE can be induced in different species by administration of myelin proteins, including myelin oligodendrocyte glycoprotein (MOG) (Evron et al., 1984, Keith, 1978). The spinal cord of EAE mice presents low expression of the central myelin proteins MOG, myelin basic protein (MBP) and proteolipid protein (PLP). Neuropathology includes loss of axons, neuronal dysfunction, infiltration of inflammatory cells, microglial activation, astrocytosis, proliferation of oligodendrocyte precursor cells, and neurological deficits (Ayers et al., 2004, Garay et al., 2005, Kim et al., 2006, McQualter and Bernard, 2007, Sun et al., 2003).
A major contributing factor to clinical symptoms of MS and EAE is the axonal damage that worsens with disease progression (Blamire et al., 2007, Dutta et al., 2007, Rovaris et al., 2005, Onuki et al., 2001, Wujek et al., 2002). Axonal pathology includes swelling, deficits in transport, degeneration and disruption of the normal axonal cytoarchitecture, changes of neurofilament phosphorylation, sodium channel distribution and development of amyloid precursor protein (APP) immunostaining (Ayers et al., 2004, Kim et al., 2006, Herrero-Herranz et al., 2008, Papadopoulos et al., 2006; Penkova and Hidalgo, 2003). There is a strong belief that restoration of myelin sheaths in EAE—a factor of critical importance for MS—will greatly preserve axonal survival (Zawadzka and Franklin, 2007). Therefore, testing neuroprotective agents that could preserve axonal integrity of EAE mice may be of potential applications in humans.
There is substantial background supporting a role for progesterone as a regulator of myelin protein expression and prevention of axonal damage both in the peripheral as well as the central nervous system (Azcoitia et al., 2003, De Nicola et al., 2006, Melcangi et al., 2005; Stein et al., 2008). When given to ovariectomized female and male animals, progesterone-treated rats show reduced axonal injury (seen via diminished APP immunoreactivity) when compared to controls (O'Connor et al., 2007). Therefore, it seems that under pathological circumstances or hormone deprivation, demyelination and axonal damage can be prevented by treatment or application of progesterone and/or its reduced derivatives.
Progesterone also induces strong immunosuppression during human pregnancy that may prevent relapses of MS (Confavreux et al., 1998, Druckmann and Druckmann, 2005, Hughes, 2004). This beneficial effect of pregnancy extends to several rodents species (Evron et al., 1984, Keith, 1978). Based on these demonstrations, the European multicentric trial POPART-MUS is currently enrolling post-partum women with MS who receive a mixture of estrogen/progestin to simulate steroid levels achieved during pregnancy, in an attempt to avoid the incidence of post-partum relapses (Confavreux et al., 1998, El-Etr et al., 2005). This clinical trial is supported by demonstrations that estrogens and progesterone protected and/or attenuate EAE development (Bebo et al., 2001, Elloso et al., 2005, Garay et al., 2007, Garay et al., 2008; Kim et al., 2006; Offner, 2004).
Previous studies have shown that progesterone pre-treatment of EAE mice attenuated the clinical outcome, inflammatory cell infiltration and demyelination of the spinal cord (Garay et al., 2007, Garay et al., 2008). Here we studied if progesterone pre-treatment also prevented axonal pathology. Our experimental approach intended to simulate the inhibitory effects of high progestin levels of pregnancy on relapses of MS and the prevention of post-partum relapses by steroid administration.
Section snippets
Results
Toluidine-blue staining of spinal cord sections showed that axonal pathology was a prominent feature of mice with induced EAE, as expected from early reports (Ayers et al., 2004, Kim et al., 2006, Papadopoulos et al., 2006; Penkova and Hidalgo, 2003). Fig. 1 (upper photomicrographs) compared a typical control mouse (a), EAE mouse (b) and EAE mouse receiving progesterone (c). A prominent axonal loss occurred in EAE mice, which showed areas of reduced staining intensity, shrunken axons and
Discussion
The present investigation shows that progesterone pre-treatment enhanced axonal density, decreased axonal damage and prevented GAP43 hyperexpression in the spinal cord of EAE female mice. The use of females was based on demonstrations that immune reactivity is greater in females than males and that men are less susceptible to the disease than women (Bebo et al., 2001, Confavreux et al., 1998, El-Etr et al., 2005). Our paradigm simulates the situation in pregnant women with MS, in which relapses
EAE induction
Adult female C57Bl/6 mice were immunized using a MOG40–54 peptide (200 μg per mouse) emulsified in complete Freund's adjuvant (Sigma, St Louis. MO) containing 0.6 mg Mycobacterium tuberculosis (Instituto Malbran, Argentina). The animals also received i.p. injections of Pertussis toxin (400 ng) (Sigma, St Louis) immediately after the immunization and another boost on the day after. Control mice received the emulsion without the MOG40–54 peptide and the Pertussis toxin. The animals developed EAE
Acknowledgments
We thank the following institutions for financial support: FONCYT (PICT 2004 25610 and PICT 2007-01044), the National Research Council of Argentina (CONICET, PIP 5542 and PIP 112-200801-00542), and the University of Buenos Aires (M016). The assistance of Mariana Lopez Ravasio with the preparation of semithin sections is gratefully acknowledged.
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