Deer antler innervation and regeneration

Abstract

Nervous system injuries are a major cause of impairment in the human society. Up to now, clinical approaches have failed to adequately restore function following nervous system damage. The regenerative cycle of deer antlers may provide basic information on mechanisms underlying nervous system regeneration. The present contribution reviews the actual knowledge on the antler innervation and the factors responsible for its regeneration and fast growth. Growing antlers are profusely innervated by sensory fibers from the trigeminal nerve, which regenerate every year reaching elongation rates up to 2 cm a day. Antler nerves grow through the velvet in close association to blood vessels. This environment is rich in growth promoting molecules capable of inducing and guiding neurite outgrowth of rat sensory neurons in vitro. Conversely, endocrine regulation failed to show effects on neurite outgrowth in vitro, in spite of including hormones of known promoting effects on axon growth. Additional studies are needed to analyze unexplored factors promoting on growth in antlers such as electric potentials or mechanical stretch, as well as on the survival of antler innervating neurons.

Full text

[Frontiers in Bioscience 17, 1389-1401, January 1, 2012]
1389
Deer antler innervation and regeneration
Manuel Nieto-Diaz1, Daniel Wolfgang Pita-Thomas2, Teresa Munoz-Galdeano1, Cayetana Martinez-Maza3, Rosa
Navarro-Ruiz1, David Reigada1, Monica Yunta1, Marcos Javier Caballero-Lopez1, Manuel Nieto-Sampedro4, Rodrigo
Martinez-Maza1
1Molecular Neuroprotection Group, Hospital Nacional de Paraplejicos (SESCAM), Finca la Peraleda s/n, 45071 Toledo (Spain),
2Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL 33136 (USA), 3Museo Nacional de
Ciencias Naturales (CSIC), C/ Jose Gutierrez Abascal 2, 28006 Madrid, Spain, 4Neural Plasticity Group, Instituto Cajal de
Neurobiologia (CSIC), Avda. Doctor Arce 37, 28002 Madrid, Spain
TABLE OF CONTENTS
1. Abstract
2. Introduction
2.1. Nervous system injuries
2.2. Therapeutic potential of the antler’s nerve regeneration
3. Antler innervation
3.1. Antler nerve supply
3.2. Nerve fiber distribution in the antler
3.3. Types of antler nerve fibers
4. Antler regulators of axon regeneration
4.1. Paracrine regulation
4.2 Endocrine regulation and other promoting mechanisms
5. Summary and perspectives
6. Acknowledgments
7. References
1. ABSTRACT
Nervous system injuries are a major cause of
impairment in the human society. Up to now, clinical
approaches have failed to adequately restore function
following nervous system damage. The regenerative cycle
of deer antlers may provide basic information on
mechanisms underlying nervous system regeneration. The
present contribution reviews the actual knowledge on the
antler innervation and the factors responsible for its
regeneration and fast growth. Growing antlers are profusely
innervated by sensory fibers from the trigeminal nerve,
which regenerate every year reaching elongation rates up to
2 cm a day. Antler nerves grow through the velvet in close
association to blood vessels. This environment is rich in
growth promoting molecules capable of inducing and
guiding neurite outgrowth of rat sensory neurons in vitro.
Conversely, endocrine regulation failed to show effects on
neurite outgrowth in vitro, in spite of including hormones
of known promoting effects on axon growth. Additional
studies are needed to analyze unexplored factors promoting
on growth in antlers such as electric potentials or
mechanical stretch, as well as on the survival of antler
innervating neurons.
2. INTRODUCTION
2.1. Nervous system injuries
Accidents causing spinal cord injury, traumatic
brain injury, stroke, or peripheral nerve injuries prevent the
correct functioning of the nervous system, causing total or
partial loss of sensory, motor, autonomic or cognitive
functions. Nervous system deficits are a major cause of
disability in developed countries. It has been estimated that
every year in the United States, 1.4 million people sustain a
traumatic brain injury (1), and 12,000 spinal cord injury (2)
while the incidence of nerve injury affects each year 13.9
per 100,000 persons in Sweden (3). In most cases, resulting
deficits are permanent. Less than 40 percent of the patients
suffering spinal cord injury (2) or 60 percent of those with
different peripheral nerve lesions (4) return to work after
injury. The social consequences are enormous since,
contrary to other pathologies like cancer or cardiovascular
diseases, neurological injuries frequently affect people
under 45 years of age, and the medical and social care is
required for decades.
Functional deficits following nervous system
damage are the direct consequence of the interruption of

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Figure 1. Illustration of the processes, regenerative capacities and functional outcomes that experience innervation following
peripheral nervous system (PNS) and central nervous system (CNS) injuries compared to those taking place during deer antler
regeneration. For each system, direct effects of damage are listed on top, below are the subsequent processes followed by the
regenerative capabilities of the system and the resulting functional outcome.
neural connections, largely due to the interruption of axons
(axotomy) and the death of neural cells (5) but also due to
inflammation, ischemia, and other processes that result in
extended cell death and disconnection (6-7, Figure 1).
Moreover, the reaction to damage of the nervous system may
also alter the neural circuitry and cause undesirable side effects
like neuropathic pain (5). Prognostic of nervous system injuries
depends on many parameters, including type, location or
extension of the lesion, age, etc. Functional deficits resulting
from severe injuries become permanent due to the limited
regeneration capacity of the nervous system. The high
differentiation degree of some neural cells, particularly neurons
or oligodendrocytes, prevents their proliferation and
replacement (8). The neurons surviving axotomy assume a
regenerative phenotype (9-11) but effective axon growth
depends on local environmental factors (see reviews in 12-13).
Mature central nervous system (CNS) environment is
inhibitory for axon growth (11,13), with few exceptions like
the olfactory or hippocampal tracts (see, for example, 14).
Adult CNS neurons also seem to present intrinsic properties
that reduce their regeneration capabilities (15). On the other
hand, peripheral nervous system (PNS) will regenerate within
the permissive growth environment of the Schwann cells (12),
although it progressively fails to sustain regenerative
response with time after injury (12). Thus, prolonged
denervations and axotomies result in poor functional
recoveries in most cases (16-17). Functional recovery
following injury is also made difficult by the high
specificity that neurons exhibit, which, in practice, means
that any new connections may not result in the recovery of
original circuits but the formation of new aberrant ones
(18).
2.2. Therapeutic potential of the antler’s nerve
regeneration
Despite more than a century of neurological
research and surgical innovation, clinical approaches have
failed to adequately restore function following central or
even peripheral nervous system damage (7). The scientific
and clinical communities have realized that the
development of efficient therapeutic tools depends on our
understanding of the damaged nervous system and our
capacity to manipulate regeneration (19). Much work has
dealt with developmental mechanisms leading to the
formation of the nervous system assuming that their
reactivation may overcome the regrowth limitations after
injury. Less attention has been paid to systems that can
regenerate spontaneously even though these systems may
provide basic information on the mechanisms that rule
nervous system regeneration (20).

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The capability to regenerate large sections of the body plan
is typical of some invertebrates and urodele amphibians
(21-23). Among adult mammals, full organ regeneration is
exceptional, being restricted to deer antlers (24). Every
year, male deers shed (cast) their antlers and fulfill a
complete regeneration process that leads to the formation of
a new set of antlers. Antler regeneration cycle has been
recognized as a valuable model to study the mechanisms
underlying organ regeneration and rapid tissue growth in
mammals (25). The whole growth period takes place in about
3 months, reaching growth rates above 2 cm a day to build up
structures of more than 3 Kg (25), up to a 20 percent of the
whole skeleton weight (26). The growing antler is an extension
of the antler pedicle periostium (27) that proliferates and
differentiates into cartilage and bone tissue to form the bone
core of the new antlers. Growing antlers are enveloped in a
hair-covered skin known as velvet that presents several
peculiarities, including lack of sweat glands and arrector pili
muscles and the presence of abundant multilobullated
sebaceous glands (28). At the end of the summer, antlers
become calcified and velvet sheds, leaving the dead bony core
used in agonistic encounters during the rut season (29). Every
year, antler innervation regenerates to provide the antler with
nerve supply (30-33). This growth supposes an extraordinary
enlargement of the peripheral field that likely arises as a local
extension of the nerve fibers that supply the forehead and the
pedicle. Once antlers stop growing and become mineralized,
nerve fibers at the velvet die back to the pedicle where they
apparently remain encapsulated (34). By the end of the winter,
when dry antlers are cast and new ones begin to grow, nerves
reenter into a regenerative state to supply the velvet antler.
Deer antlers are a very interesting source of
information on the mechanisms underlying the nerve
regeneration and functional recovery following injury
(20,23,35). This spontaneous regeneration model is
particularly interesting for therapeutic studies because it
occurs in an adult mammal and involves cells, mechanisms
and/or biochemical pathways which are more likely to be
similar to those in humans than other regenerating models
as the non-mammalian vertebrates (newts or fishes) or
invertebrates. Moreover, antler regeneration takes place in
adult individuals and affects adult neural cells like those
typically involved in nerve injuries and different from those
of embryos with different growth capabilities (see for
example, 15, 36). The extraordinary extension and growth
rate that the nerve fibers achieve during antler regeneration
is particularly outstanding from both the clinical and
biological points of view (Figure 1). Gray and colleagues
(32) demonstrated that nerve supply grows together with all
antler tissues to cover all antler (see also 33,37,38),
reaching elongation rates over 2 to 3 cm a day in the largest
species like the moose (Alces alces; 24) or the wapiti
(Cervus canadenisis; 39). From the biological point of
view, it is particularly remarkable the transport rate and the
amounts of cell material needed to enlarge axons more than
one meter at rates above 2 cm per day, especially if we
consider that maximum axon growth by growth-cone
extension only reaches 1 mm per day (40-41). In fact, such
an extremely fast growth is not consistent with current
understanding of the transport of essential structural
elements such as neurofilament proteins, for which the
average transport speed is limited to 1 mm per day (42-45).
The rapid and sustained nerve growth observed in the
antlers indicates that the physiological capacity of axons to
expand rapidly and continuously is not limited by protein
synthesis, transport rates, or the availability of structural
constituents (43-44,46-48). The clinical consequences of
understanding this phenomenon would be enormous,
considering that in many injuries, growing axons have to
reach far away targets, up to one meter in the case of some
human nerve fibers. Reconnecting these targets would take
years for axons growing at elongation rates around 1 mm
per day, resulting in functional recovery failure (17).
However, at the rate observed in deer antlers, it would take
a few months of growth.
3. ANTLER INNERVATION
3.1. Antler nerve supply
The first description on the anatomy of the deer
antler innervation dates back to the 19th century (49). Later
studies on Virginia deer (Odocoileus virginianus; 30), red
deer (Cervus elaphus, 50), wapiti (cervus canadensis, 51),
and fallow deer (Dama dama, 51) showed that the nerves
supplying the antlers come from the supraoptic
(infratrochlear) branch of the ophtalmic division and the
zygomaticotemporal branch of the maxillary division, both
from the trigeminal (5th cranial) nerve (Figure 2). The
supraoptic branch is a single bundle that emerges from the
skull beneath the upper edge of the orbit (1.5-2 cm from the
medial canthus of the eye in red deer according to Adams,
50) and courses medially over the dorsal rim, near the
medial angle of the eye (51). It then courses caudally
through the orbicularis oculi muscle and sends several
nerves toward the base of the antlers where 6 or more small
branches in Virginia deer (30) or a full web in wapiti (51)
are given off to the anterior and medial surfaces of the
pedicle and the antler. The zygomaticotemporal branch of
the maxillary nerve emerges onto the scalp as a large nerve
near the zygomatic arch, at the caudal margin of the
zygomatic process of the frontal bone, and divides
immediately, producing a number of branches coursing
toward the antler base and the ear. The branches that supply
the antler pass caudodorsally through the retrorbital
(periorbital) fat and beneath the frontalis muscle. Midway
from the orbit to the pedicle they branch into several nerves
(6 or more in Virginia deer according to 30), which
disperse on the lateral and posterior surfaces of the antler
pedicle and then onto the antler. Both trigeminal nerve
supplies follow very closely the respective distribution of
the lateral and medial coronary branches of the superficial
temporal artery (the last branch of the external carotid) that
provides the antler blood supply (30,51). Besides the
trigeminal innervation, fibers from the zygomatic branch of
the auriculopalpebral nerve (a branch of the facial or 7th
cranial nerve) have been observed to reach the medial
surface of the pedicle (50-51) although there is no evidence
of its extension into the antler. According to Woodbory and
Haigh (51), no other nerves could be traced to the pedicle.
3.2. Nerve fiber distribution in the antler
Several authors have studied the location of the
nerve supply in the growing antler (30-32,34,37-38).

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Figure 2. Illustration detailing the antler nerve supply.
Growing antlers are innervated by the zygomaticotemporal
and supraoptic branches of the trigeminal nerve (solid
black lines) that reach the antler accompanying the
superficial temporal artery (in gray dashed lines).
Growing antlers consist on tips of densely packed
mesenchymal cells which differentiate proximally into
cartilage and bone tissue (Figure 3). Overlaying cartilage
and bone, there is a layer of perichondrium/periostium that
ultimately shows continuity with the mesenchyme
primordia. The entire antler is covered by velvet, a
modified skin with abundant hair follicles and sebaceous
glands but without arrector pili muscles and sweat glands
(28). Histologically, velvet consists on a thick epidermis
without invaginations that covers a thick dermis. Velvet
dermis can be subdivided into an outer dermis, in contact
with the epidermis, that contains numerous hair follicles
and sebaceous glands, and an inner dermis, located
beneath, that does not present any visible structure. Beneath
the dermis appears a layer of highly-vascularized loose
connective tissue -termed vascular layer by several authors
(30-31,33-34,52)- that separates the tegument from the
avascular mesenchyme. Wislocki and Singer (30) used
Bodian's protargol method to show profuse nerve fibers
occurring in small bundles at the vascular layer in Virginia
deer. Similar results were obtained by Vacek (31) for red
deer, fallow deer and roe deer (Capreolus capreolus), and
Li et al. (34) for red deer, specifying that nerves passed
through and above the vascular stratum of the velvet from
where they projected to more superficial layers.
Immunohistochemical analyses in red deers by Gray and
colleagues (30) showed that innervation concentrate on
deep connective layers of the velvet, which anatomically
correspond to the vascular layer. They also observed nerve
fibers in the dermis, the epidermis the
periostium/perichondrium and even in cartilage tissues
within the central core of the antler. In 2007, Li et al. (37)
also observed nerve fibers immunoreactive to the 200 Kda
neurofilament (NF200), traveling through the mesenchyme
primordium. In order to examine antler nerve distribution
in detail, we used antibodies against different
neurofilaments to stain antler sections confirming that, in
agreement with previous studies, most fibers are located
deep in the velvet, mainly between the dermis and the
vascular layer (33,38,53, Figure 3). Most fibers appear
isolated at the antler tip while they usually form bundles at
the base. Nerve branches project from the vascular layer
towards more superficial layers of the velvet but, contrary
to Li et al. (37) and Gray et al. (32), we have not observed
them in the mesenchyme or derived tissues (33,38,53).
Antler nerve fibers are both myelinated and not-myelinated
(30-31) as confirmed by the presence of glial fibrillary
acidic protein (GFAP) immunoreactive Schwann cells
accompanying or enseathing axons in the velvet tissues
from the base to the tip of the antler (Figure 3).
3.3. Types of antler nerve fibers
Antler nerve fibers show specific
immunoreactivity to markers of distinct sensory neuron
populations (32-33,37-38,53). Primary sensory fibers can
be characterized by their neurochemical signature, in
particular their immunoreactivity to CGRP (calcitonin gene
related peptide), Substance P, and NF200 peptides and
isolectin B4 (IB4) from Griffonia simplicifolia (54-56).
According to this classification, myelinated Abeta fibers
are only immunoreactive to NF200, the thin myelinated
Adelta fibers are immunoreactive to both NF200 and
CGRP; while the un-myelinated peptidergic C fibers are
immunoreactive to the nociceptive peptides CGRP and
substance P. Finally, non-peptidergic C fibers are marked by
IB4. Gray and colleagues (32) observed peptidergic C fibers
immunoreactive to substance P and CGRP surrounding blood
vessels and as free fibers. Li and colleagues (37) showed the
presence of nerve fibers immunoreactive to the NF200 in the
antler. Recently, we have combined immunohistochemistry for
NF200 and CGRP to carry out a detailed analysis of the antler
innervation, from the trigeminal or semilunar ganglion to the
antler tip (38). Stained sections of the trigeminal nerve showed
the presence of bundles of NF200 positive, CGRP negative
fibers, others with fibers positive for both markers and some
fibers exclusively marked against CGRP (Figure 4). The same
combinations of markers were observed at the vascular layer of
the antler, mostly as bundles in the base and as free fibers at
the tip, although bundles were occasionally seen at the tip and
free fibers at the base (38,53). However, although we could
establish the presence of fibrous structures IB4 positive, the
lack of colocalization with axon markers and the fact that IB4
also stains vessels and even Schwann cells precludes the
unequivocal confirmation of the presence of these fibers in the
antler. Antler nerve fibers seem to end freely in the tissue,
lacking specialized sensory nerve receptors (30-31). Free fiber
endings are characteristic of mechanoreceptors of Abeta fibers,
and the majority of Adelta and C fibers. Abeta fibers convey
touch information from skin receptors to mechanical
stimuli of low intensity (cutaneous mechanoreceptors),
while Adelta fibers that function as receptors for pressure,
touch and cold temperature as well as convey fast pain
information from acute noxious stimuli, and peptidergic C
fibers, which are warmth and slow pain receptors (also
responsible for itch sensation). In the antler, both Abeta and
Adelta fibers would provide precise localization of the
stimulus or damage location, being responsible for the
withdrawal reflex and the extreme sensitivity to touch
stimuli of the growing antlers (37). On the contrary, C
fibers would cause slow “burning” pain and release CGRP
and Substance P which are involved in inflammation and
wound healing.
Besides sensory innervation, antlers do not seem
to present other nerve components. Motor fibers have not
been observed in agreement with the lack of muscles in the

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Figure 3. Anatomy of the antler innervation. A) Histology of the antler tip (hematoxilin eosin) specifying its constituting tissues.
B) Detail of the antler tip (hematoxilin eosin). E=epidermis, OD=outer dermis, ID=inner dermis, VL=vascular layer,
M=mesenchyme, PC=precartilague. C) Corresponding serial section showing nerve fibers (green) immunostained with antibody
against Neurofilament and cell nuclei stained with Hoechst (blue). Axons are located over the vascular layer (arrow), but also at
the dermis (star) or within the vascular layer (asterisk). D) Schwann cells (GFAP, red) enseathing an axon (Neurofilament
panclonal, green) at the antler tip.
antler. Sympathetic innervation of the blood vessels was
suggested by Vacek (31), who described nerve fibers in
the adventitia and the media of the antler arteries.
However, neither previous anatomical studies by
Wislocki and Singer (30) nor later studies by Rayner
and Ewen (57) and Gray et al. (32) have confirmed the
presence adrenergic nerve fibers in the blood vessels of
any part of the antler, although they were evident at the
pedicle (57). In fact, description, antler arteries seem to
be constructed to close themselves by constriction in the
event of being severed, being independent from innervation
(30).
4. ANTLER REGULATORS OF AXON
REGENERATION
4.1. Paracrine regulation
The factors underlying nerve fiber growth in the
antler remain largely unknown. However, a paracrine
regulation can be expected, due to the presence in the antler

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Table 1. Axon growth promoters identified in the growing
antler
Trophic factors
Neurotrophins
• Nerve Growth Factor
• Neuritrophin-3
• Brain Derived Growth Factor
Epitelial growth factors
• Epidermal Growth Factor
Fibroblast growth factors
• basic Fibroblast Growth Factor
Insulin-like growth factors
• Insulin-like Growth Factor-1
• Insulin-like Growth Factor-2
Transforming growth factor family
• Bone Morphogenetic Protein 2
• Bone Morphogenetic Protein 3B
• Bone Morphogenetic Protein 4
• Transforming Growth Factor beta
Vascular endotelial growth factor
Neurite growth promoting factors
• Midkine
• Pleiotrophin
Serpins
• Pigment Epitelium Derived Factor
Other factors
• Glucose-6-Phosphate Isomerase
• Meteorin
• Retinoic acid
Extracellular matrix molecules
• Laminin
• Collagen I
Glycosaminoglycans
• Heparan sulfate
Cell adhesion molecules
• Neuronal Cell Adhesion Molecule
For references see 57
of Schwann cells and endothelial cells which constitute a
normal source of neural growth promoters. In agreement,
antlers express nerve growth factor (NGF, 37) and
neurotrophin 3 (NT3, 56), two well-known axon growth
factors of the neurotrophin family. Recently, we combined
different molecular techniques to determine the gene
expression of axon growth promoters in the antler velvet
(59). Microarray analysis allowed us to hypothesize the
expression or change in expression of 90 promoters or
regulators of the axonal growth. 15 of them were sequenced
and analyzed by semiquantitative RT-PCR, establishing the
expression in the antler velvet of brain derived neurotrophic
factor (BDNF), glucose phosphate isomerase (GPI),
meteorin (MTRN), midkine (MDK), and neuronal cell
adhesion molecule (NRCAM), not previously observed in
deer. Combining these data with previous analyses, a list of
more than 20 axon growth promoters can be obtained
drawing a picture of the growth promoting environment of
the antler innervation during its annual regeneration. The
list (Table 1) comprises several neurotrophins and growth
factors, such as basic fibroblast growth factor (FGFb),
epidermal growth factor (EGF), pleiotrophin, or pigment
epithelium derived factor (PEDF), morphogens of the
transforming growth factor (TGFbeta) family, members of
the insulin-like growth factor (IGF) family, together with
retinoic acid, and several substrate molecules like collagen,
laminin and heparan sulphate. All these molecules have
demonstrated direct or indirect axon growth promoting
properties for different neuronal types, including sensory
neurons like those innervating the antler, either in culture or
in vivo. Most of them also promote axon regeneration
following nervous system damage (60-67) or have been
identified in models of epimorphic regeneration, like in the
newt limbs, or in the fish fins. Classic among them are
retinoic acid (68-69), bone morphogenetic proteins (BMPs,
70-71) or FGFb (69-70,72-73), but also collagen (70,74),
heparan sulphates (75), laminin (76), IGF (77) or TGFbeta
(78). In most cases, these molecules are necessary for the
organ regeneration to be completed or even initiated, but
little is known on their roles in the axon growth during the
epimorphic regeneration process. Table 1 also shows that
most factors are present or expressed in the velvet skin,
where nerves are located, except for the BMPs. In fact,
immunohistochemical and in situ hybridization studies
indicate the presence or expression of most promoters in
the inner vascular layer of the velvet dermis where most
nerve fibers are observed. This is the case of NGF (37),
pleiotrophin (79), EGF (52), FGFb (80), and vascular
endothelial growth factor (VEGF, 80). Such spatial co-
localization indicates a direct exposure of the axons to
these molecules and would confirm a relevant role for them
in antler nerve growth, either promoting axon regrowth or
meeting the trophic requirements of the axons. However,
comparisons with published information on individual
molecules or from human gene expression profiles stored in
the GeneNote database (81) reveals that all identified
promoters are also expressed in normal or wounded skin.
Moreover, comparison of the gene expression between the
antler velvet and the skin overlaying the antler pedicle or
the frontal bone confirms this similarity (59). The only
exception is Midkine, which is significantly overexpressed
in the velvet respect to frontal skin samples, though not
respect to pedicle skin. Somehow, Midkine expression
profile fits with what can be expected from a molecule
involved in the process of rapid axonal growth in the antler.
The capabilities of the antler molecules to
promote neurite growth were first evaluated by Huo and
colleagues (82), who observed neurite outgrowth from
PC12 cells cultured with growing antler tissue extract. We
further analyzed the axon growth promoting properties of
the different tissues of the growing antler (33). The analysis
of the effects of antler extracellular matrix and cell
adhesion molecules on neurite outgrowth from dorsal root
ganglia (DRG) demonstrated that the antler vascular layer
has the capability to guide but not to promote axon growth.
These results suggest the action of axonal guidance
systems, in agreement with a strong orientation of the
collagen fibers in deep velvet layers (83) or the
predominant expression of NGF in the vascular layer of the
dermis (37). On the other hand, soluble molecules secreted
by the antler tip velvet promote a significant increase in
neuritogenesis and neurite outgrowth in DRG neurons from
rat embryos (33), trigeminal neurons from adult rats (53)
and PC12 cells (in preparation), as illustrated by Figure 5.
Biochemical treatments using enzymatic digestion, heat
denaturation, and size filtering treatments proved that the
neurite outgrowth promoters are most likely proteins.
Experiments using blocking antibodies (33,53) showed that
NGF blockage caused a significant reduction of neurite

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Figure 4. Fiber types of the antler innervation according to their neurochemical signature. A to D: control staining of the
ophthalmic nerve at the exit of the trigeminal ganglion. A and B show fibers immunostained against NF200 (green) and CGRP
(red) with all possible combinations of the markers (NF200+/CGRP-, NF200+/CGRP+, and NF200-/CGRP+). C and D show the
staining of isolectin B4 (green) and the axon marker panclonal neurofilament. No NFp+ fibers appear marked by IB4. Some IB4+
cells surrounding NFp+ axons (arrows in D) could correspond to Schwann cells. E and F show different combinations of CGRP
(red) and NF200 (green) immunoreactivity in nerve fibers at the base (E) and the tip (F) of the antler. All possible combinations
are onserved, corresponding to Abeta (NF200+/CGRP-), Adelta (NF200+/CGRP+) and peptidergic C fibers (NF200-/CGRP+).
Nerve bunches are observed at both the base (E) and the tip (F) but they are clearly larger at the base.
growth, proving a clear contribution of NGF to the effect of
the velvet in agreement with its expression in vivo (37).
However, velvet medium still kept significant promoting
effects, indicating that other proteins also contributed to
these growth properties.
4.2. Endocrine regulation and other promoting
mechanisms
Antler regeneration cycle is strongly regulated by
hormones (84). Some hormones with axon growth
promoting properties like IGF-1 (85-86) or triiodothyronine
(T3; 87) show high blood levels during the antler growth
period (88-90). However, in vitro analyses indicate that the
neurite growth promoting properties of serum obtained
during antler maximal growth do not differ from those of
serum from other antler cycle stages (33). It is intriguing
why strong differences in the serum concentration of IGF-1
during the antler regeneration cycle (84) are not reflected
by differences in neurite growth in these assays. It is
possible that the increased number of Schwann cells caused
by the deer sera would mask any direct effect of serum
molecules on neurite outgrowth. However, that would point
to a very modest promoting effect of the endocrine
regulators and therefore, they could not be leading
participants of the high growth rates registered in antler
axons. Nevertheless, it is also possible that molecules with
higher levels in serum during antler regeneration could
influence indirectly axon growth, for example, by acting on
the expression of neurotrophic factors or even acting in
synergy with other factors.

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Figure 5. Neurite outgrowth induced by soluble molecules secreted by the antler velvet. Illustrative images showing the
differences in neurite outgrowth of adult rat trigeminal neurons (upper row), rat embryo DRG neurons (middle row), and PC12
line cells (lower row) cultured with basal medium (negative control, left column), medium supplemented with NGF (center
column), and conditioned (supplemented) by antler velvet tissue (right column).
In addition to paracrine and endocrine regulation,
other less-classical factors may contribute to cause the very
fast axonal growth observed in the deer antlers, including
mechanical stretch or electric fields. Growing antlers present
negative electrical potentials between their tip and base, which
vary in marked correlation with the growth rate of the antler
(91). Similar electric potentials have been observed to promote
and orientate axonal growth during development and
regeneration (see 92 for review). The influence of electric
fields on antler nerve growth was already proposed by Bubenik
(23,93), who also proposed that electric stimulation of the
growth of the nerve branches innervating the antler would also
induce a general growth of the antler, both in size and
complexity. Parallel evidences also support the contribution of
mechanical stretch to the antler nerve growth. Antler velvet
experience strong mechanical stretch due to the fast growth
of the underlying mesenchyme (83). According to Li and
Suttie (28), this stretch may be responsible for the growth
and properties of the antler velvet. As shown in different in
vitro studies, such tensions can induce higher axon growth
rates than any other promoter (94-95). In fact, mechanical
stretch seems to be responsible for the axon elongation
during postnatal development, when axons have already
connected to their targets but still have to grow (95).
5. SUMMARY AND PERSPECTIVES
We have just begun to analyze the axon
regeneration in the deer antler (33,37,53,58,59,95). The
analyses have shown that the antler velvet provides the
growing axons with a environment rich in promoting
factors, with NGF having a basic role (33,37,82,96),
although other paracrine regulators should also contribute
(33). In this respect, our group is using proteomic
techniques to isolate and identify axon growth promoters
secreted by the velvet. Preliminary results have identified
more than 80 secreted or membrane proteins with
documented relationships to axon growth, including cell
adhesion, extracellular matrix and soluble molecules.
However, it is also necessary to evaluate the effect of
unexplored factors like mechanical stretch, or electric
fields, which may also contribute or even determine the
growing characteristics of the antler innervation.
Despite the potential of these approaches,
understanding the nerve regeneration process and its
regulators would greatly benefit from the direct study of the
antler neurons. The study of the antler’s transcriptome,
particularly the analysis of the trigeminal neurons

Deer antler innervation
1397
innervating the antler, would be also very helpful to
identify and characterize the processes taking place during
the antler regeneration. This approach, followed by other
cell and molecular analyses, would allow the
characterization of these neurons and the pathways
activated during the antler regeneration. The recent
sequencing and annotation of the red deer genome by
researchers from New Zealand and the development of the
High Throughput Sequencing methodologies capable of
sequencing hundreds of millions RNA molecules have opened
the opportunity to carry out these analyses. The resulting
dataset would help many research groups working on this
model as well as researchers from related fields to access
detailed information on all tissues involved in the process at
different times.
Besides nerve regeneration, functional repair of
injured nervous system also depends on protecting nerve cells
from cell death. Apoptotic cell death following CNS injury is
responsible for a significant loss of functional capabilities (97-
98). Even in the peripheral nervous system, 20 to 50 percent
of sensory neurons die through apoptosis or similar
processes during the weeks following injury (6,7,99-100),
and some specific populations almost completely disappear
(100). The total number of dying cells varies depending on
factors like the age, the location of the injury or its extent
(7). Apoptosis of axotomized neurons may be triggered by
antidromic electrical activity or neurotoxic inflammatory
agents, or most likely by the subsequent loss of target-
derived neurotrophic support (101). Conversely, the
sensory neurons supplying the growing antler do not seem
to experience relevant cell death processes even 8 months
after axotomy. Although direct evidence of neuron survival
or death after antler mineralization is still lacking, the
presence of encapsulated axon ends at the pedicle (34)
suggests that neurons innervating the antler survive
axotomy and regenerate as collaterals from these
encapsulated fibers to supply the new antler one year later.
6. AKNOWLEDGEMENTS
The present contribution was supported by
funding from the Health Department of the Castilla-La
Mancha government (projects ICS-06025 and PI2008-38).
We are deeply in debt to Andres García, Tomas Landete,
Laureano Gallego from IREC and UCLM, and Javier
Martin from VenissonDeer for providing the deer samples
used in our studies. We also thank an anonimous reviewer
for his/her helful comments on the manuscript.
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Abbreviations: CNS central nervous system, PNS
peripheral nervous system, NF200 200Kda neurofilament,
CGRP Calcitonin-gene related peptide, IB4 isolecting B4,
GFAP glial fibrillary acidic protein, NGF nerve growth
factor, NT-3 neurotrophin 3, BDNF brain derived
neurotrophic factor, GPI glucose phosphate isomerase,
MTRN meteorin, MDK midkine, NRCAM neuronal cell
adhesion molecule, FGFb basic fibroblast growth factor,
EGF epidermal growth factor, PEDF pigment epithelium
derived factor, TGFbeta transforming growth factor beta,
BMP bone morphogenetic protein, IGF insulin-like growth
factor, VEGF vascular endothelial growth factor, DRG
dorsal root ganglia.
Key Words Deer antler, Velvet, Endocrine, Paracrine
regulation, Fast axon growth, Trigeminal nerve, Nervous
system injury, Spontaneous regeneration, Neuron, Gene
expression, in vitro analysis
Send correspondence to: Manuel Nieto-Diaz, Molecular
Neuroprotection Group, Hospital Nacional de Paraplejicos
(SESCAM), Finca La Peraleda s/n. 45071 Toledo, Spain,
Tel: 34 925396834, Fax: 34 925247745, E-mail:
mnietod@sescam.jccm.es
http://www.bioscience.org/current/vol17.htm