Factors Promoting Neurite Outgrowth During Deer Antler Regeneration Wolfgang Pita-Thomas, 1,2 * Manuel Nieto-Sampedro, 1,2 Rodrigo M. Maza, 2 and Manuel Nieto-Diaz 1,2 1 Experimental Neurology Unit, Hospital Nacional de Paraple ´jicos (SESCAM), Toledo, Spain 2 Neural Plasticity Department, Instituto Cajal de Neurociencias (CSIC), Madrid, Spain Every year male deers completely regenerate their ant- lers. During this process, antlers are reinnervated by sensory fibers, growing at the highest rate recorded for any adult mammal. Despite its clinical potential, only a few studies have dealt with this fascinating phenom- enon. Among the possible factors underlying fast growth of the antler’s innervation, the effects of the antler’s en- docrine and paracrine factors were evaluated, using an in vitro assay for sensory neurite growth. We found that soluble molecules secreted by the velvet, the modified skin that covers the antler, strongly promote neurite out- growth. Using specific blocking antibodies, we demon- strated that nerve growth factor is partially responsible for these effects, although other unidentified molecules are also involved. On the contrary, neither endocrine se- rum factors nor antler substrates promoted neurite out- growth, although antler substrata from deep velvet layers cause neurite outgrowth orientation. Taken to- gether, our results point to the existence in the deep vel- vet of an environment that promotes oriented axon growth, in agreement with the distribution of the antler innervation. V V C 2010 Wiley-Liss, Inc. Key words: dorsal root ganglion (DRG); velvet conditioned medium; cryoculture; axonal growth; endocrine regulation Adult mammals have a very limited capability for organ regeneration. However, every year males from all deer species (Cervidae, Artiodactyla) cast their antlers and fulfill a complete regeneration process leading to the formation of a new set of antlers. To achieve antler regeneration, mesenchymal cells proliferate at the antler tip and differentiate distally into chondroblasts, chondro- cytes, and later osteoblasts and osteocytes (Price et al., 2005). During this process, osteocartilaginous tissue is enveloped by velvet, a modified skin characterized by a lack of sweat glands and errector pili muscles and the presence of abundant multilobullated sebaceous glands (Li and Suttie, 2000). Both osteocartilaginous tissue and velvet grow in the red deer (Cervus elaphus) to reach over 1 meter of length in approximately 3 months (Goss, 1970). During their regeneration, antlers are in- nervated by myelinated and unmyelinated sensory fibers of the trigeminal nerve that travel through the velvet (Wislocki and Singer, 1946; Gray et al., 1992). Nerve fibers grow together with other antler tissues, reaching growth rates over 2 cm per day in the largest species (Gray et al., 1992), the highest axonal growth rate regis- tered in adult mammals. Only a few studies have dealt with the regeneration and fast growth of the antler’s innervation, but several underlying mechanisms and processes can be hypothe- sized. Endocrine participation can be expected, insofar as the antler regeneration cycle is strongly regulated by hormones (Bubenik, 1990), some of which, such as in- sulin-like growth factor 1 (IGF-1) and triiodothyronine (T3), are well-known axonal growth promoters (Schenker et al., 2002; Rabinovsky, 2004) and are pres- ent at high levels during the period of antler growth. In addition, paracrine regulation can be expected also, as suggested by the presence of well-characterized neuro- trophic factors, such as nerve growth factor (NGF; Li et al., 2007), neurotrophin-3 (NT-3; Garcia et al., 1997), epidermal growth factor (EGF; Barling et al., 2005), vascular endothelial growth factor (VEGF), pleio- trophin (Clark et al., 2006), and bone morphogenetic proteins (BMPs; Feng et al., 1995, 1997; Kapanen et al., 2002), together with axon growth-promoting extracellu- lar matrix molecules (ECMs), such as laminin (Korpos et al., 2005) and heparan sulfate proteoglicans (Ha et al., 2005). Other growth-promoting factors, such as me- chanical stretch (Smith, 2009) or electric fields (McCaig et al., 2002), have been also described for the antlers during their annual renewal (for electric fields see, for example, Lake et al., 1979; for mechanical stretch see Li and Suttie, 2000, and references therein). Contract grant sponsor: ICS; Contract grant number: 06025; Contract grant sponsor: FISCAM (to W.P.T.); Contract grant number: PI-2008/ 38; Contract grant sponsor: Health Council of the JCCM (Spain); Con- tract grant sponsor: Ministry of Science and Technology of Spain (to W.P.-T.). *Correspondence to: Dr. Wolfgang Pita-Thomas, Experimental Neurol- ogy Unit, Hospital Nacional de Paraple ´jicos (SESCAM), Finca la Pera- leda s/n. 45071, Toledo, Spain. E-mail: dwpita@sescam.jccm.es Received 1 February 2010; Revised 28 April 2010; Accepted 29 April 2010 Published online 13 July 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jnr.22459 Journal of Neuroscience Research 88:3034–3047 (2010) ' 2010 Wiley-Liss, Inc.