NEUROPROTECTIVE EFFECTS OF DIHYDROPROGESTERONE AND PROGESTERONE IN AN EXPERIMENTAL MODEL OF NERVE CRUSH INJURY I. ROGLIO, a R. BIANCHI, b S. GOTTI, c S. SCURATI, d S. GIATTI, a M. PESARESI, a D. CARUSO, d G. C. PANZICA c,e AND R. C. MELCANGI a * a Department of Endocrinology and Center of Excellence on Neurode- generative Diseases, University of Milano, Via Balzaretti 9, 20133 Milano, Italy b Department of Molecular Biochemistry and Pharmacology, “Mario Negri” Institute for Pharmacological Research, Via Eritrea 62, 20157 Milano, Italy c Laboratory of Neuroendocrinology, Department of Anatomy, Pharma- cology and Forensic Medicine, Neuroscience Institute of Torino, Uni- versity of Torino, C.so M. D’Azeglio 52, 10126 Torino, Italy d Department of Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133 Milano, Italy e Istituto Nazionale di Neuroscienze, (INN), Italy Abstract—A satisfactory management to ensure a full resto- ration of peripheral nerve after trauma is not yet available. Using an experimental protocol, in which crush injury was applied 1 cm above the bifurcation of the rat sciatic nerve for 20 s, we here demonstrate that the levels of neuroactive ste- roids, such as pregnenolone and progesterone (P) metabolites (i.e. dihydroprogesterone, DHP, and tetrahydroprogesterone, THP) present in injured sciatic nerve were significantly de- creased. On this basis, we have focused our attention on DHP and its direct precursor, P, analyzing whether these two neu- roactive steroids may have neuroprotective effects on bio- chemical, functional and morphological alterations occurring during crush-induced degeneration-regeneration. We dem- onstrate that DHP and/or P counteract biochemical alter- ations (i.e. myelin proteins and Na  ,K  -ATPase pump) and stimulate reelin gene expression. These two neuroactive ste- roids also counteract nociception impairment, and DHP treat- ment significantly decreases the up-regulation of myelinated fibers’ density occurring in crushed animals. Altogether, these observations suggest that DHP and P (i.e. two neuro- active steroids interacting with progesterone receptor) may be considered protective agents in case of nerve crush injury. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: rat sciatic nerve, neuroactive steroids, myelin proteins, reelin, morphology, nociception. Peripheral nerves are frequently exposed to physical injury and this can result in a severe functional impairment and decreased quality of life because of sensory and motor loss of function. In the last decade, several therapeutic approaches have been developed to stimulate the regeneration of the nerve, such as the administration of neurotrophins (Terenghi, 1999; Boyd and Gordon, 2003; Gordon et al., 2003), of extracellular matrix molecules (Woolley et al., 1990; Labrador et al., 1998), or the application of electrical stimulation (Nix and Hopf, 1983; Al-Majed et al., 2000). Unfortunately, all these methods are limited in scope be- cause they do not ensure a full restoration of function. Therefore, new strategies that simultaneously potentiate axonal regeneration, promote remyelination and the recov- ery of nerve functions are needed. Recent observations obtained in experimental models of peripheral neuropathy, such as diabetes, aging, hered- itary neuropathy, etc., indicate that neuroactive steroids exert protective effects (Azcoitia et al., 2003; Sereda et al., 2003; Melcangi and Garcia-Segura, 2006; Leonelli et al., 2007; Roglio et al., 2007, 2008). Interestingly, in some of these experimental models, we demonstrated that neuro- degenerative process itself affects the levels of neuroac- tive steroids present in peripheral nerves (Roglio et al., 2007; Caruso et al., 2008a,b). In addition, studies in experimental models of periph- eral neuropathy due to physical injury demonstrated prom- ising protective effects of neuroactive steroids. For in- stance, in the experimental model of rat nerve transection, progesterone (P) and its metabolite, dihydroprogesterone (DHP), significantly increase the gene expression of a myelin protein, glycoprotein zero (P0) (Melcangi et al., 2000). After cryolesion of sciatic nerve in male mice, ad- ministration of P or its precursor, pregnenolone (PREG), to the lesion site increases the extent of myelin sheath for- mation (Koenig et al., 1995). Moreover, P-loaded chitosan prostheses produce the best-guided nerve regeneration response in rabbit facial nerve after axotomy (Chavez- Delgado et al., 2005). Finally, in peripheral nerve injury models, such as hamster facial motoneuron, rat sciatic motoneuron, rat pudendal motoneuron, etc., testosterone and its derivative, dihydrotestosterone, accelerate regen- eration and functional recovery of nerves (Yu, 1982; Vita et al., 1983; Tanzer and Jones, 1997, 2004; Jones et al., 2001; Huppenbauer et al., 2005). Nerve crush injury in rat is a commonly used axonot- metic model (i.e. an experimental model in which injury has the potential to recover and recover completely) to study *Corresponding author. Tel: +39-02-50318238; fax: +39-02-50318204. E-mail address: roberto.melcangi@unimi.it (R. C. Melcangi). Abbreviations: ANOVA, analysis of variance; Ct, cycle threshold; Cx32, connexin 32; DHP, dihydroprogesterone; MAG, myelin-associ- ated glycoprotein; MAL, myelin and lymphocyte protein; MBP, myelin basic protein; MS/MS, tandem mass spectrometry; P, progesterone; PBS, phosphate buffer solution; PCR, polymerase chain reaction; PMP22, peripheral myelin protein 22; PREG, pregnenolone; P0, gly- coprotein zero; SFI, sciatic functional index; THP, tetrahydroprogest- erone; 5-R, 5-reductase. Neuroscience 155 (2008) 673– 685 0306-4522/08$32.00+0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.06.034 673