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Experimental Neurology 259 (2014) 2837

Contents lists available at ScienceDirect

Experimental Neurology
journal homepage: www.elsevier.com/locate/yexnr

Review

Sex steroids and neuroprotection in spinal cord injury: A review of


preclinical investigations
Stella Elkabes a,, Arnaud B. Nicot b,c,d
a

The Reynolds Family Spine Laboratory, Department of Neurological Surgery, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, USA
UMR 1064, INSERM, Nantes, France
Facult de Mdecine, Universit de Nantes, France
d
ITUN, CHU de Nantes, France
b
c

a r t i c l e

i n f o

Article history:
Received 27 October 2013
Revised 25 December 2013
Accepted 4 January 2014
Available online 16 January 2014
Keywords:
Gender
Spinal cord
Trauma
Regeneration
Estrogen
Progesterone
Testosterone
Cell death
Myelination
Motor function

a b s t r a c t
Spinal cord injury (SCI) is a debilitating condition that affects motor, sensory and autonomic functions. Subsequent to the rst mechanical trauma, secondary events, which include inammation and glial activation, exacerbate tissue damage and worsen functional decits. Although these secondary injury mechanisms are amenable to
therapeutic interventions, the efcacy of current approaches is inadequate. Further investigations are necessary
to implement new therapies that can protect neural cells and attenuate some of the detrimental effects of inammation while promoting regeneration.
Studies on different animal models of SCI indicated that sex steroids, especially 17-estradiol and progesterone,
exert neuroprotective, anti-apoptotic and anti-inammatory effects, ameliorate tissue sparing and improve functional decits in SCI. As sex steroid receptors are expressed in a variety of cells including neurons, glia and immune system-related cells which inltrate the injury epicenter, sex steroids could impact multiple processes
simultaneously and in doing so, inuence the outcomes of SCI. However, the translation of these pre-clinical ndings into the clinical setting presents challenges such as the narrow therapeutic time window of sex steroid administration, the diversity of treatment regimens that have been employed in animal studies and the lack of
sufcient information regarding the persistence of the effects in chronic SCI. The current review will summarize
some of the major ndings in this eld and will discuss the challenges associated with the implementation of sex
steroids as a promising treatment in human SCI.
2014 Elsevier Inc. All rights reserved.

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Animal models of SCI, functional outcome measures and histopathological parameters
Sex steroid receptor signaling and expression in the spinal cord . . . . . . . . . .
Estrogen receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Progesterone receptors . . . . . . . . . . . . . . . . . . . . . . . . . .
Androgen receptors . . . . . . . . . . . . . . . . . . . . . . . . . . .
The effects of 17-estradiol in spinal cord injury . . . . . . . . . . . . . . . . .
Effects on functional recovery and tissue sparing . . . . . . . . . . . . . .
Anti-apoptotic and neuroprotective effects . . . . . . . . . . . . . . . . .
Effects on oligodendrocytes and myelination . . . . . . . . . . . . . . . .
Anti-inammatory effects and modulation of glial reactivity by 17-estradiol . .
The effects of progesterone in SCI . . . . . . . . . . . . . . . . . . . . . . . .
Effects on functional recovery and tissue sparing . . . . . . . . . . . . . .
Effects on neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects on glia, myelination and inammation . . . . . . . . . . . . . . . .
Androgens and spinal cord injury . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author at: Department of Neurological Surgery, NJMS-Rutgers, 205 South Orange Avenue, Cancer Center F 1204, Newark, NJ 07103, USA.
E-mail address: stella.elkabes@rutgers.edu (S. Elkabes).
0014-4886/$ see front matter 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.expneurol.2014.01.008

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S. Elkabes, A.B. Nicot / Experimental Neurology 259 (2014) 2837

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction
Traumatic spinal cord injury (SCI) is a debilitating condition that can
lead to permanent neurological disability. It is estimated that approximately 273,000 people live with SCI in the United States and the annual
incidence is about 12,000 new cases per year (NSCISCwww.uab.edu/
nscisc). Major causes of SCI include trafc and sport accidents, falls
and violence. SCI is most common among young adults with an incidence rate that is 34 times higher in male than female subjects
(DeVivo, 2012). However, increased incidence among elderly females,
primarily due to falls, has been reported (Furlan et al., 2005). In addition
to loss of locomotor function, affected individuals suffer from secondary
co-morbidities such as chronic pain, spasticity, and bladder, bowel and
sexual dysfunction.
The initial traumatic event that elicits tissue destruction in the injured spinal cord is followed by cellular and molecular changes which
occur within hours to weeks and cause further damage to the spared
tissue. The secondary events include activation of glia, inltration of
immune system-related cells, induction of inammation, secretion of
detrimental effectors that cause neuronal and oligodendrocyte death
and demyelination. The formation of a cavity and the glial scar at the
injury epicenter and the molecular milieu at the lesion site constitute
major impediments to the regeneration of transected axons and
remyelination of spared, intact axons. Yet, these secondary injury mechanisms are amenable to treatment and could be targets of therapeutic
interventions that modulate inammation, promote neuroprotection,
enhance regeneration and reinstate function. Despite many medical advances, the current therapies have limited efcacy and do not effectively
alleviate neurological decits or adequately restore function.
Studies on animal models have shown gender differences in functional outcomes of SCI, with remarkably better locomotor recovery in
female than male rodents (Farooque et al., 2006; Hauben et al., 2002).
These ndings, together with investigations reporting benecial effects
of 17-estradiol and progesterone in animal models of brain injury and
disease (for reviews see Brown et al., 2009; Deutsch et al., 2013; Herson
et al., 2009), spurred interest in the neuroprotective roles of sex steroids
in SCI (Kwon et al., 2011). Yet, controversy exists on gender-differences
in individuals with SCI. Greenwald et al. (2001) did not nd genderrelated differences in Functional Independence Measure (FIM) motor
scores or American Spinal Injury Association (ASIA) scores at acute
care admission or rehabilitation discharge. In individuals with incomplete tetraplegia, no signicant gender differences were noted in
motor and sensory neurologic recovery at the time of admission, rehabilitation discharge or during the follow-up years (Pollard and Apple,
2003). Co-morbidities, mortality, discharge disposition and neurologic
outcomes were not signicantly different in men and women with similar cervical SCI, although women showed a trend for higher incidence
of reactive depression and venous thrombosis (Furlan et al., 2005). In
contrast, a multi-center study on a large number of individuals with
SCI indicated gender differences in certain (but not all) neurologic
scores. Whereas the mean motor index scores at admission and 1 year
post-injury were not statistically different in women and men with
SCI, the degree of neurologic improvement, evaluated as the difference
between motor index scores at 1 year post-injury and at admission,
was signicantly better in women than in men sustaining complete or
incomplete SCI (Sipski et al., 2004). However, when FIM motor scores
were analyzed, in general, men functioned better than women at the
time of rehabilitation discharge. Finally, no signicant differences were
observed in the neurologic recovery of pre- and post-menopausal
women with SCI. However, a denite conclusion could not be reached

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due to the lack of information about estrogen replacement therapy in


post-menopausal women sustaining a SCI.
The aim of this review is to provide an overview of the animal
studies that assessed the effects of sex steroids in SCI. We will start
by presenting a brief description of the various animal models, the
functional outcome measures and the histopathological parameters
that are frequently used in investigations of SCI. Subsequently, we
will summarize some of the ndings related to the effects of sex
steroids in animal models of SCI. Finally, we will discuss the challenges associated with the translation of the pre-clinical studies
into the clinical setting.
Animal models of SCI, functional outcome measures and
histopathological parameters
Evaluation of therapeutic strategies that promote functional recovery following SCI necessitates the use of clinically relevant animal
models and reliable outcome measures that reproducibly assess
motor, sensory and autonomic functions. A number of methods have
been used to induce SCI in rodents (Rosenzweig and McDonald, 2004;
Onifer et al., 2007). As the majority of injuries in humans are the result
of a blunt trauma, animal models of contusive injury are increasingly
accepted as paradigms mimicking the human condition. Different
devices such as the New York University (NYU) (Gruner, 1992) or
MASCIS impactor (Young, 2002), the Ohio State University (OSU) impactor (Bresnahan et al., 1987; Noyes, 1987) and the Innite Horizon
SCI device (Rabchevsky et al., 2003; Scheff et al., 2003) are used to perform contusion injuries in rodents. Compression of the rat spinal cord
with an aneurysm clip (Fehlings and Tator, 1995; Rivlin and Tator,
1978), forceps crush injury in rodents (Plemel et al., 2008; Zhang and
Guth, 1997) and complete or incomplete transection injury, including
dorsal and lateral hemisection, have also been utilized (Onifer et al.,
2007; Rosenzweig and McDonald, 2004).
Open eld locomotor function in rats and mice that sustain thoracic
injury is often assessed by use of the Basso, Beattie and Bresnahan (BBB)
rating scale for rats (Basso et al., 1995) and the Basso Mouse Scale (BMS;
Basso et al., 2006). The modied Tarlov Scale (Gale et al., 1985), originally developed by Tarlov and Klinger (1954), has also been used to assess
motor movement in rats sustaining SCI. Additional tests have been designed to evaluate motor function in rats with cervical SCI (Martinez
et al., 2009; Webb and Muir, 2005). The CatWalk gait analysis assesses
parameters of locomotion including stride characteristics (Hamers
et al., 2006). In addition, the grid walk test, the inclined plane test
(Muir et al., 2007) and the horizontal ladder test (Soblosky et al.,
2001) assess motor or sensorimotor function. The hotplate paw withdrawal test and the Von Frey hair test are frequently used to measure
thermal and mechanical hypersensitivity, respectively (Mills et al.,
2001). At the histopathological level, the most prevalent measurements
are lesion volume and spared white or gray matter (Basso, 2004). Tissue
sparing has been correlated with recovery of locomotor function (Basso,
2004).
Sex steroid receptor signaling and expression in the spinal cord
In this review, we will focus on actions of sex steroids which are
achieved by plasma levels encountered in males or females during
physiological/supra-physiological situations including stress and
pregnancy: concentrations below 0.1 M for estrogens or androgens
and below 1 M for progesterone (for a review see Nicot, 2009). Sex steroids exert both genomic and non-genomic actions through receptors

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S. Elkabes, A.B. Nicot / Experimental Neurology 259 (2014) 2837

that are located in the nucleus and membranes. These receptors are
expressed in different cell types of the central nervous system (CNS)
and modulate cell signaling and gene expression via various mechanisms. To better understand how sex steroids might affect the course
and outcomes of SCI, it is necessary to dene the cellular distribution of
sex steroid receptors in the spinal cord, the signaling pathways that
they utilize and the cellular processes that they inuence.
Estrogen receptors
Estrogen exerts its genomic or non-genomic actions via two traditional estrogen receptors ESR1/ER and ESR2/ER (Nilsson and
Gustafsson, 2002). More recently, an additional new estrogen receptor,
G protein-coupled estrogen receptor (GPER-1/GPR30, currently referred to as GPER) has been identied (Revankar et al., 2005). This
membrane-bound receptor has been implicated in the mediation of
non-genomic effects of estrogen.
The genomic actions of estrogen are initiated upon binding ESR1 and
ESR2 in the cytoplasm or nucleus. Liganded ESR1 or ESR2 can directly
regulate transcriptional processes in the nucleus, either by binding specic estrogen response elements (EREs), which are located within the
promoters of target genes, or by interacting with other DNA-binding
transcription factors that modulate gene expression. The target genes include anti-apoptotic mediators of the bcl-2 family and pro-inammatory
cytokines.
These receptors can also be associated with the plasma membrane
and mediate some of the non-genomic actions of estrogen, including
regulation of gene expression through second messengers, modulation
of protein kinase activity and regulation of ionic ux, including calcium
ux (Razandi et al., 1999; Vasudevan and Pfaff, 2008; Wade et al., 2001).
In the brain, estrogen-induced neuroprotection is mediated by
membrane-associated ER signaling (Mannella and Brinton, 2006).
ESR1 and ESR2 are also found in the mitochondria where they
regulate the expression containing genes (Stirone et al., 2005; Yager
and Chen, 2007; Yang et al., 2004). Interestingly, estrogen receptordependent signaling via mitogen activated protein kinase (MAPK) and
nuclear factor kappa B (NFB) pathways results in an upregulation of
antioxidant enzymes (Borras et al., 2007). This pathway may indirectly
mediate the antioxidant effects of estrogens which could be important
in neuroprotection.
Another important estrogen receptor involved in rapid signaling
is GPER, which is localized to both the endoplasmic reticulum and
the plasma membrane. This receptor can mediate some of the estrogen
effects that were previously attributed to ESR/ERs (Prossnitz and Barton,
2011). Selective ESR1/ER, ESR2/ER and GPER/GPR30 agonists and
antagonists are available and facilitate studies on estrogen receptors,
although the further development of more selective compounds is
warranted (Carroll et al., 2012; Dennis et al. 2011; Oyola et al., 2012).
In the uninjured spinal cord, estrogen receptors are found in both
neurons and different glial subtypes (Papka et al., 2001). Several spinal
cord neuronal populations express low to moderate levels of ESR1 or
ESR2, whereas spinal cord astrocytes mostly express ESR1/ER
(Giraud et al., 2010) and spinal cord microglia primarily express ESR2/
ER (Wu et al., 2013). All estrogen receptor subtypes are found in oligodendrocyte progenitors and oligodendrocytes, in vitro (Jung-Testas
et al., 1992). However, ER is the main isoform expressed in oligodendrocytes of the adult CNS including the spinal cord and is localized to
the plasma membrane and myelin (Arvanitis et al., 2004). GPER/
GPR30 is also found in dorsal and ventral horn neurons (Dun et al.,
2009; Hu et al., 2012) as well as oligodendrocytes and microglia
(Hirahara et al., 2013; Hu et al., 2012). In addition to the spinal cord,
GPER/GPR30 mRNA and protein have been detected in neurons of the
dorsal root ganglia (DRG). These neurons project to the spinal cord dorsal horns and convey sensory information. In accordance, GPER/GPR30
immunoreactivity is localized to the supercial layers of the dorsal
horns and dorsal rhizotomy induces the transport of this receptor

from the cell bodies in the DRG to axon terminals in the dorsal horns
(Takanami et al. 2010).
Following SCI, immune system-related cells inltrate the injury
epicenter. These cells could also be the target of estrogen as studies
have shown expression of estrogen receptors in immune cells inltrating
the inamed spinal cord in animal models of multiple sclerosis (Giraud
et al., 2010; Wu et al., 2013). With regard to GPER/GPR30, its relatively
high expression in rodent and human lymphoid tissues suggests that
the receptor might be involved in the regulation of immune cell function
(O'Dowd et al., 1998; Owman et al., 1996). In agreement with this idea, a
GPER/GPR30 agonist reduced endotoxin-elicited inammatory mediator
production by macrophages (Rettew et al., 2010) and modulated antiinammatory functions of T cells (Brunsing et al., 2013).
Progesterone receptors
As in the case of estrogen, progesterone exerts genomic and nongenomic actions, directly via the nuclear progesterone receptor (PR,
with PR-A isoform and the N-terminal extended isoform PR-B) or indirectly, at the plasma membrane level (Schumacher et al., 2007). In addition, new membrane progesterone receptors and progesterone binding
proteins, that are not related to PR-A/B, have been described. For example, progesterone can bind neuronal 1 receptors and modulate glutamate signaling while the progesterone metabolite, allopregnanolone,
is a potent modulator of the GABA type A receptor (Faroni and
Magnaghi, 2011; Schumacher et al., 2007). Such interactions may be
important in the control of spinal pain processing (Aouad et al., 2013;
Juif et al., 2013; Patte-Mensah et al., 2014).
The expression of progesterone receptors PR-A is relatively low
in the CNS. In the rodent spinal cord, PR expression was found in both
neurons and astroglial cells (Labombarda et al., 2003, 2010).
Androgen receptors
Androgens, including testosterone and the more active metabolite,
5-dihydrotestosterone (DHT), are known to exert their effects through
the activation of intracellular receptors that regulate the transcription
of target genes. Two isoforms of the classical AR have been described
(AR-A and its N-terminally extended form, AR-B) in different cell types.
The existence of a plasma membrane receptor for androgens has also
been proposed. Classical genomic and nongenomic mechanisms, including the activation of signaling pathways, such as the MAPK pathway,
have been associated with these receptors (Nicot, 2009).
It is worth noting that aromatase, which is expressed by various cell
types including brain cells, can convert testosterone, but not the more
potent metabolite DHT, into 17-estradiol (Cherrier et al., 2005). The
DHT metabolite 5-androstane-3, 17-diol (3Adiol) can also directly
activate ER (Pak et al., 2005). Thus, testosterone metabolites, produced
in vivo, have the potential to act on ERs or GPER.
In addition to the well-known high AR expression in specic hypothalamic nuclei and brain regions regulating reproduction, subsets of
dorsal horn neurons and motoneurons show substantial AR expression
(Lumbroso et al., 1996; Shughrue et al., 1997; Simerly et al., 1990).
The effects of 17-estradiol in spinal cord injury
Effects on functional recovery and tissue sparing
A number of studies reported benecial effects of 17-estradiol on
functional outcomes of SCI. Recovery of locomotor function was the
most frequent outcome measure assessed. Yune et al. (2004) showed
a signicant improvement in BBB scores when male rats, that sustained
a mild contusion injury at mid-thoracic level, received a single intravenous (i.v.) injection of a low 17-estradiol dose (100 g/kg) either
12 h before injury or 1 h post-injury. Amelioration of BBB scores was
paralleled by a reduction in lesion volume at 24 weeks post-injury.

S. Elkabes, A.B. Nicot / Experimental Neurology 259 (2014) 2837

Lee et al. (2012) used a similar injury paradigm and found that male rats
that received 17-estradiol at 6 and 24 h post-injury had higher BBB
scores and performed better in the inclined plane test, grid walk test
and footprint analysis at 34 weeks post-injury. However, if the rst
17-estradiol delivery was delayed until 12 h post-injury, no improvement in functional decits occurred.
Amelioration of motor skills was also reported when 17-estradiol
pellets were implanted subcutaneously to male rats sustaining a cervical hemicontusion injury (Siriphorn et al., 2012). The pellets slowly
release 17-estradiol within the course of 21 days and sustain high
17-estradiol blood levels. As early as 2 weeks post-injury, 17estradiol-treated injured rats performed signicantly better than
vehicle-treated injured rats in skilled forelimb function, assessed by
the modied vermicelli handling test (Allred et al., 2008). Amelioration
of skilled locomotor function occurred at 7 weeks post-injury. Histological analyses revealed a signicantly higher preservation of white matter in 17-estradiol treated rats.
Investigations were also performed on young (pre-menopausal)
and old (post-menopausal) female ovariectomized (OVX) rats that
were implanted with Silastic capsules containing either 180 g/ml
17-estradiol or vehicle, prior to a crush injury at the mid-thoracic
level. 17-Estradiol-treated, young OVX rats had signicantly better
BBB scores compared to vehicle-treated OVX or normally cycling rats
(Chaovipoch et al., 2006). However, there were no signicant differences between the BBB scores of vehicle-treated OVX rats and normally
cycling rats. This indicates that endogenous estrogen does not inuence
recovery of locomotor function and exogenous 17-estradiol is necessary to observe amelioration of motor decits. Accordingly, signicantly
more white matter was spared when OVX rats received exogenous 17estradiol, whereas endogenous estrogen did not alter the extent of
white matter sparing. Using a similar paradigm, favorable but less pronounced effects of 17-estradiol were also observed in older rats.
Additional studies that employed different rat or mouse SCI models
and distinct 17-estradiol treatment regimens indicated improvement
of locomotor function (Cuzzocrea et al., 2008; Ritz and Hausmann,
2008; Sribnick et al., 2010). In contrast to all the reports mentioned
above, Swartz et al. (2007) did not detect any alterations in BBB scores
when female OVX rats or male rats that sustained a mid-thoracic contusion injury were treated with 17-estradiol delivered in Silastic capsules (180 g/ml or 1 mg/ml) implanted 7 days prior to injury.
An interesting investigation addressed whether endogenous androgens alter the benecial effects of 17-estradiol on locomotor function
(Kachadroka et al., 2010). These studies compared the effects of 17estradiol on gonadectomized and intact male rats after a mid-thoracic
crush injury. The results indicated that testicular-derived endogenous
androgens do not alter the benecial effects of 17-estradiol.
Information about the effects of estrogen on autonomic function following SCI is scarce. 17-Estradiol attenuated autonomic dysreexia in
mice sustaining a complete transection injury (Webb et al., 2006), whereas no effects on bladder function were observed in crush injured OVX rats
(Chaovipoch et al., 2006). With respect to central neuropathic pain,
Hubscher et al. (2010) reported favorable effects of endogenous sex steroids when at-level pain responses were compared in cycling and OVX
injured rats. Injured male rats treated with 17-estradiol also showed
an improvement in pain-related responses (Hubscher et al., 2010).
In an attempt to explain the mechanisms underlying the effects of
17-estradiol, the aforementioned studies and a number of other investigations explored the cellular and molecular changes that occur in the
injured spinal cord following treatment. Attenuation of apoptosis,
reduction in neuronal and oligodendrocyte loss and a decrease in inammation and reactive gliosis were the most frequent observations.
Anti-apoptotic and neuroprotective effects
Evidence indicates that 17-estradiol exerts anti-apoptotic and neuroprotective effects in rodents sustaining SCI. 17-Estradiol reduced the

31

number of TUNEL positive cells at the epicenter when treatment was


initiated before or immediately after injury (Cuzzocrea et al., 2008;
Schmitt et al., 2006; Sribnick et al., 2006; Yune et al., 2004). In addition,
neuronal apoptosis appeared diminished when treatment with a low
17-estradiol dose was initiated at 4 h post-injury, although no stereological quantication was available in this report (Samantaray et al.,
2011). In accordance with these ndings, the injury-elicited increase
in the activity of caspase-3 and caspase-9, proteases mediating apoptotic cell death, was reduced in the spinal cord of rats treated with 17estradiol prior to or immediately after induction of SCI (Lee et al.,
2012; Yune et al., 2004). Increased expression of anti-apoptotic genes
bcl-2 and bcl-x and decreased expression of the pro-apoptotic gene
bax were observed in 17-estradiol treated injured mice and rats
(Cuzzocrea et al., 2008; Samantaray et al., 2011; Yune et al., 2004).
The changes in bax and bcl-2 expression extended into the sub-acute
phase in injured male rats, when 17-estradiol treatment was initiated
immediately following injury and continued for 6 weeks (Sribnick et al.,
2010). Ventral horn neuron number was higher in cycling and 17estradiol-treated OVX rats sustaining SCI, than injured, OVX rats receiving vehicle. This indicates that both endogenous 17-estradiol and
exogenous 17-estradiol enhance neuronal survival after SCI
(Chaovipoch et al., 2006). In the injured spinal cord, the benecial effects of 17-estradiol on apoptosis can be mimicked by a GPER agonist
and are abolished when GPER expression is knocked down (Hu et al.,
2012). Thus, with respect to apoptosis, GPER could be the principal
mediator of 17-estradiol actions in SCI.
Effects on oligodendrocytes and myelination
The anti-apoptotic effects of 17-estradiol in SCI are not conned to
neurons. In male rats sustaining a contusion injury, repeated administration of 17-estradiol reduced oligodendrocyte apoptosis (Lee et al.,
2012). It is likely that these protective effects are exerted, at least partly,
through direct actions on oligodendrocytes. This idea is supported by
in vitro investigations showing that the death of cortical oligodendrocytes, elicited by a cytotoxic agent, is reduced by 17-estradiol (Takao
et al., 2004). In addition, studies indicate effects on myelin formation.
17-Estradiol delays cell cycle exit of oligodendrocyte progenitors and
enhances myelin sheet formation, in vitro (Marin-Husstege et al.,
2004). This nding is in accordance with an early report showing
stimulatory effects of 17-estradiol on myelination, during development (Curry and Heim, 1966). In a preclinical setting, 17-estradiol
together with progesterone prevents partially cuprizone-induced
demyelination (Acs et al., 2009). The estrogen receptor (ER) ligand diarylpropionitrile (DPN), administered after disease onset,
has remyelinating/neuroprotective effects in a murine model of multiple sclerosis (Khalaj et al., 2013). A pro-myelinating effect of GPER/
GPR30 signaling has also been demonstrated, in vitro, whereas a
GPER/GPR30 agonist improved re-myelination after cuprizoneelicited demyelination, in vivo (Hirahara et al., 2013). Additional investigations are necessary to assess whether 17-estradiol supports
the re-myelination of spared, demyelinated axons following SCI.
Anti-inammatory effects and modulation of glial reactivity by 17-estradiol
A number of reports show that the SCI-elicited inltration of monocytes, macrophages and neutrophils is reduced following 17-estradiol
treatment. A high 17-estradiol dose, administered to male rats immediately after SCI, caused a signicant decrease in the number of inltrating
cells at the injury epicenter and the caudal penumbra (Sribnick et al.,
2005). Other investigations conrmed the reduction in the spreading of
inammatory cells and the decrease in the number or activity of
macrophages/microglia in the injured spinal cord (Ritz and Hausmann,
2008; Samantaray et al., 2011; Siriphorn et al., 2012). The attenuation of
microglia/macrophage reactivity, assessed qualitatively, appeared to
continue even in chronic phases (Sribnick et al., 2010).

32

S. Elkabes, A.B. Nicot / Experimental Neurology 259 (2014) 2837

Limited information is available regarding the effects of 17estradiol on activated astrocytes in SCI. Studies were conducted in different animal models using distinct treatment strategies and outcomes
were evaluated at different time points post-injury. Ritz and Hausmann
(2008) found that a high 17-estradiol dose, delivered immediately
after injury, enhances the SCI-induced increase in GFAP and vimentin
immunoreactivity in rat astroglia. In injured rats treated with 17estradiol for 21 days, reactive astrogliosis, evaluated by measuring
GFAP immunoreactivity, is attenuated at 50 days post-injury, a time
when there is resolution of inammation (Siriphorn et al., 2012). Reactive gliosis can also be downregulated by testosterone but this effect has
been largely attributed to its conversion into estradiol (Garcia-Estrada
et al., 1993).
With regard to pro-inammatory cytokine and chemokine expression, Ritz and Hausmann (2008) found that 17-estradiol increases
further the injury-elicited, transient upregulation of IL-1 and IL-1
protein content in the spinal cord at 6 h post-injury. However, 17estradiol treatment decreased the SCI-provoked increase in TNF-,
IL-1 and CCL2/MCP-1 mRNA levels in mice at 24 h post-injury
(Cuzzocrea et al., 2008). Estrogen, acting via ESR2/ER and/or GPER/
GPR30, prevents transcription of inammatory cytokine genes in
microglia/macrophages by interfering with the translocation of NFB
from the cytoplasm to the nucleus (Ghisletti et al., 2005; Hu et al.,
2012; Wu et al., 2013). Estrogen also dampens proinammatory
chemokine gene expression in spinal cord astrocytes, an effect involving
direct interaction of ESR1 with NFB in the nucleus (Giraud et al., 2010).
Such mechanisms may be important in SCI, especially in view of investigations showing that astrocyte-selective inhibition of NFB reduces
proinammatory cytokine and chemokine expression and improves
locomotor function and tissue sparing following contusive SCI (BracchiRicard et al., 2013; Brambilla et al., 2005).
The effects of progesterone in SCI
Effects on functional recovery and tissue sparing
The effects of progesterone on locomotor decits and tissue sparing
following SCI remain controversial. An early study on male rats sustaining a mid-thoracic contusion injury indicated a signicant improvement
in BBB scores at six weeks post-injury, when rats were treated for
5 days with a high progesterone dose (4 mg/kg, i.p.) starting immediately after injury (Thomas et al., 1999). This was paralleled by a signicant preservation of the white matter. In contrast, when male and
female rats, which received a moderate mid-thoracic contusion injury,
were treated for 5 days with 4 mg/kg or 8 mg/kg progesterone, no improvement in BBB scores or tissue sparing was observed (Fee et al.,
2007). Administration of a higher progesterone dose for a longer period
of time was not effective either. However, in male rats sustaining a
hemi-section, daily subcutaneous injection of 16 mg/kg progesterone
prevented the development of mechanical allodynia and attenuated
the injury-provoked cold sensitivity (Coronel et al., 2011).
Effects on neurons
The majority of the studies that investigated the effects of progesterone on neurons in SCI, used hemisection or total transection models and
a 3-day course of progesterone (4 mg/kg) treatment that started shortly
after injury. Choline acetyl transferase immunoreactivity, which is decreased in ventral horn neurons following SCI, is restored by progesterone treatment (Labombarda et al., 2002). In addition, progesterone
enhanced the expression of GAP43, a protein involved in axonal elongation. The expression of brain derived neurotrophic factor (BDNF) is
reduced in ventral horn neurons following SCI, but reinstated after
progesterone administration to injured rats (Gonzalez et al., 2005).
The neuroprotective effects of progesterone were corroborated by
a study reporting a reduction in neurons undergoing SCI-provoked

chromatolysis following progesterone-treatment (Gonzalez et al.,


2009). In addition, the injury-induced decrease in microtubuleassociated protein 2 (MAP2), an essential element of the cytoskeleton,
returned to control values following progesterone administration
(Gonzalez et al., 2009). Because the investigations cited above did not
include assessment of locomotor, sensory or autonomic functions, it is
not clear whether the progesterone-induced neuronal changes correlate with improvement of neurologic decits. It would be valuable to
address this issue in future investigations.
Effects on glia, myelination and inammation
In a complete transection SCI model in male rats, a 3-day progesterone regimen (4 mg/kg), starting shortly after injury, increased NG2 positive (NG2 +) oligodendrocyte progenitor cell number (Labombarda
et al., 2006, 2009). A later study by the same group used an identical
model but a higher progesterone dose (16 mg/kg) for a short (3 days)
or longer (21 days) treatment period (Labombarda et al., 2011). The
short treatment induced oligodendrocyte progenitor cell proliferation
whereas the longer-term treatment promoted the differentiation of
NG2+ oligodendrocyte progenitors into oligodendrocytes. These ndings, combined with an earlier report showing an increase in proteolipid
protein after administration of a similar progesterone regimen to rats
with SCI (Labombarda et al., 2009), suggested that long-term progesterone treatment promotes differentiation of oligodendrocyte progenitors
into mature oligodendrocytes (Labombarda et al., 2011). Although progesterone increases oligodendroglial cell maturation and promotes
remyelination (Schumacher et al., 2007), it is not yet known whether
this holds true in SCI.
Progesterone also impacts reactive gliosis and microglial activation
following SCI (Labombarda et al., 2011). In agreement with these ndings, progesterone exerts anti-inammatory effects on astrocytes and
microglia in vitro (e.g., regulation of inducible nitric oxide synthase
expression; Nicot, 2009). Such effects may be mediated by PR-A/B or
unrelated receptors that may also play a role in the control of pain at
the spinal cord level (Faroni and Magnaghi, 2011; Patte-Mensah et al.,
2014).
In male rats sustaining a compression injury, administration of
medroxyprogesterone acetate (MPA), a synthetic progestin, shortly
after SCI, reduced the injury-provoked increase in the levels of
malonyldialdehyde, a marker of lipid peroxidation (Topsakal et al.,
2002) and of TNF- (Sahin et al., 2011) at the injury epicenter.
Androgens and spinal cord injury
A considerable number of men living with SCI have low serum testosterone levels (Bauman et al., 2011; 2014; Celik et al., 2007; Clark
et al., 2008a; Durga et al., 2011). Despite the higher incidence of SCI in
men, little is known about the impact of testosterone deciency on neural damage following SCI. Yet, early studies have shown that testosterone protects spinal cord neurons against excitotoxic cell death, in vitro
(Ogata et al., 1993). In animal models of motor neuron or nerve injury,
benecial effects of exogenous testosterone administration have also
been reported (Fargo and Sengelaub, 2004a,b; Tehranipour and
Moghimi, 2010). Androgens prevent the axotomy-induced death of
spinal motor neurons during development (Gould et al., 1999). Implantation of testosterone-containing capsules to female rats sustaining a
mid-thoracic contusion injury improves SCI-induced abnormalities in
motoneuron and muscle morphology but does not alter lesion volume,
tissue sparing or the number of motoneurons (Byers et al., 2012). A
potential benecial effect of testosterone replacement therapy on
motor function in men with incomplete SCI has been reported although
it was not clear whether this was due to an improvement in muscle
strength or neuroprotection (Clark et al., 2008b).
However, it is important to note that testosterone has also been implicated in detrimental effects in vitro or in other models of CNS injury.

S. Elkabes, A.B. Nicot / Experimental Neurology 259 (2014) 2837

33

locomotor function in female rats compared to males has been reported


(Farooque et al., 2006; Hauben et al., 2002). However, it is not clear
whether there is a gender difference in the clinical outcomes of SCI.
What are the challenges of translating 17-estradiol therapy into a
clinical setting for the treatment of SCI? First, it is difcult to compare
the pre-clinical investigations performed in different laboratories because of the diversity of SCI paradigms and rodent species/strains
used, and the distinct 17-estradiol treatment regimens implemented
(Table 1). Continuous administration of 17-estradiol by means of subcutaneously implanted pellets or Silastic capsules, a single injection of
17-estradiol following SCI or repeated injections over hours or days
are dissimilar treatment strategies that can impact in different ways
the injury-provoked mechanisms and outcomes. Another major challenge is the time window of 17-estradiol administration. In the majority of animal studies, treatment was initiated prior to or immediately
after SCI, a time frame that is not applicable to human SCI. Even a 4 to
6 hour delay in the initiation of treatment could be difcult to implement in clinics. Very few studies examined the outcomes of SCI when
17-estradiol was administered at 12 h post-injury or later. A low
17-estradiol dose was ineffective when treatment was initiated at
12 h post-injury, indicating a narrow therapeutic time window. On
the other hand, the high 17-estradiol doses used in some reports
raise concerns about the use of such treatments, considering health
benets versus risks in humans. Moreover, most investigations focused
on acute or subacute phases and less information is available about the
persistence of the effects in chronic phases. Finally, locomotor recovery
was the most frequently assessed functional outcome measure after
17-estradiol treatment, whereas autonomic and sensory functions
were infrequently evaluated. This is especially important in view of a
report indicating that a GPER agonist induces mechanical hyperalgesia

Although activation of the classical intracellular/nuclear AR generally


promotes MAPK- and phosphoinositide 3-kinase-dependent neuroprotection, cortical astrocytes express a plasma membrane AR that
promotes cell death. This may explain the brain damaging effects of testosterone treatment in a rodent model of stroke (Gatson and Singh,
2007). In addition, even though only few oligodendrocyte subsets
express AR in the CNS (Lorenz et al., 2005), in the presence of an aromatase inhibitor, testosterone amplies excitotoxic damage to oligodendrocytes, in vitro (Caruso et al., 2004). In contrast, a recent report
showed that testosterone or the safer androgenic drug, 7-methyl-19
nortestosterone, promotes myelin repair in the CNS via androgen
receptor signaling in cells of the neural lineage (Hussain et al., 2013).
Androgen receptor signaling may also have effects on activated microglia, and possibly on astrocyte subsets, in specic CNS regions (Nicot,
2009). These controversial issues have not yet been resolved in the
context of SCI.
Discussion
Despite the diversity of SCI paradigms and the dissimilar dosage
and treatment regimens used (summarized in Table 1), the majority
of animal studies reported a benecial effect of 17-estradiol on recovery of locomotor function and tissue sparing, which corresponded with
attenuation of inammation and apoptosis following SCI. However, it
is important to point out that controversy exists, as one study could
not nd effects of 17-estradiol neither on functional outcomes nor histopathological, cellular and molecular parameters. The severity of the
injury, the rat or mouse strains and sub-strains used and other experimental conditions could be the source of this disparity (Kjell et al.,
2013; Popovich et al., 1997; Schmitt et al., 2006). A better recovery of

Table 1
Summary of SCI paradigms, 17-estradiol treatment regimen and outcome measures in studies on rats and mice.
SCI paradigm

T10 contusion injury in


male rats
T12 contusion injury in
male rats
T12 contusion injury in
male rats
T8T9 crush injury in
female, OVX rats
T10 contusion injury in
male or OVX female
rats
T8T9 compression
injury in male rats
T10 contusion injury in
male rats
T8T9 crush injury in
gonadectomized and
intact male rats
T8 contusion injury in
male and female OVX
or cycling rats
T10 contusion injury in
male rats

17-Estradiol regimen

Reference

Time of administration

Functional tests

Histological/molecular analyses

100 g/kg, i.v.

12 h prior to SCI or 1 h p.i.

BBB

Lesion volume, apoptotic markers

Yune et al. (2004)

4 mg/kg, i.v.

15 min followed by 24 h p.i.

None

Sribnick et al. (2005)

4 mg/kg, i.v.

15 min followed by 24 h p.i.

None

Silastic capsules,
180 g/ml, s.c.
Silastic capsules,
180 g/l or 1 mg/ml,
s.c.
0.1 or 4 mg/kg, i.p.

Implanted 1 week prior to SCI

BBB, bladder function

Implanted 7 days prior to SCI

BBB

White matter sparing, markers of


inammation.
Apoptotic markers, calpain
activation
White matter sparing,
neurodegeneration, apoptosis
Lesion length, total tissue sparing

Immediately p.i.

BBB, narrow beam


crossing test
BBB

Lesion volume, markers of


Ritz and Hausmann (2008)
inammation, gliosis
Markers of inammation, apoptotic Sribnick et al. (2010)
markers, white matter sparing

BBB

White matter sparing, neuronal


survival apoptotic markers

Kachadroka et al. (2010)

4 mg/kg, i.v. followed


by 2 mg/kg, i.p.
Pellets, 0.055 mg/kg,
s.c.

15 min and 24 h p.i. (i.v.)


followed by daily i.p. injections
for 5 days
30 min p.i.

Sribnick et al. (2006)


Chaovipoch et al. (2006)
Swartz et al. (2007)

Pellets, 0.25 mg, s.c.

One week prior to SCI

Semmes-Weinstein
monolament test

White matter sparing

Hubscher et al. (2010)

110 g/kg i.v. followed


by 110 g/kg/day via
osmotic pumps s.c.
10 g/kg
300 g/kg, i.v.

Immediately p.i. or 15 min,


2 h, 4 h p.i.

None

Microgliosis, apoptotic markers

Samantaray et al. (2011)

BBB, inclined plane, grid


walk, footprint analysis
BBB
Vermicelli handling test;
horizontal ladder test
CatWalk gait analysis
Autonomic dysreexia

Apoptotic markers, axonal loss,


oligodendrocyte death
Apoptosis
Motor neuron survival, white
matter sparing, reactive gliosis.

Lee et al. (2012)

T10 contusion injury in


male rats
T9 contusion in male rats 100 g/kg, i.v.
C5 hemi-contusion in
Pellets, 0.5 or 5 mg, s.c.
male rats
T2 complete transection
in male mice
T6T7 compression
injury in male mice

Major outcome measures

Dose/route

Pellets, 0.5 mg
implanted, s.c.
300 g/kg, s.c.

15 min and 24 h p.i


5 min followed by 6 and
24 h p.i. or 12 h p.i.
15 min followed by 24 p.i.
30 min p.i.

One week prior to SCI


1 h before injury followed
by 3 and 6 h p.i.

Modied BBB

Hu et al. (2012)
Siriphorn et al. (2012)

Lesion length, Microglial reaction

Webb et al. (2006)

Inammatory and apoptotic


markers

Cuzzocrea et al. (2008)

34

S. Elkabes, A.B. Nicot / Experimental Neurology 259 (2014) 2837

by acting on a subgroup of DRG nociceptive neurons, which project to


the dorsal horns (Kuhn et al., 2008). Another investigation showed
that intrathecal administration of a GPER agonist promotes painrelated behaviors and elicits spinal nociception (Deliu et al., 2012).
Additional studies also support the notion of pronociceptive effects of
estrogens in the dorsal spinal cord (Zhang et al., 2012).
The aforementioned issues underscore the importance of validating
further the effects of 17-estradiol in pre-clinical investigations using a)
SCI models that mimic best the human condition, b) low dose 17estradiol, c) treatment regimens that are applicable to humans including the time of treatment initiation and d) time frames for the evaluation of outcome measures in chronic phases. Standardization of the
SCI animal model and the 17-estradiol regimen as well as inclusion
of tests to assess sensory and autonomic functions may provide better
insights into the potential of 17-estradiol as a therapeutic agent in
SCI. Longitudinal studies on the effects of 17-estradiol on the inammatory response, axonal regeneration, re-myelination and neuroprotection and correlation of such measures with functional outcomes are
some future directions that warrant investigations. Further exploration
of molecular and cellular mechanisms underlying 17-estradiol actions
could dene additional targets for therapeutic interventions.
With regard to progesterone, only few investigations reported functional outcomes and these results were controversial (Fee et al., 2007;
Thomas et al., 1999; Table 2). Since the two discordant studies used similar treatment regimens but different contusion models, the method
employed or the severity of injury could be factors contributing to the
discrepant outcomes. Although there is ample information about the
cellular and molecular changes induced by progesterone treatment following SCI, these studies did not assess functional outcomes concomitantly, and therefore, it is not possible to correlate the molecular/
cellular alterations with improvement of neurologic decits. Nevertheless, neuroprotective effects of progesterone have been reported in
other pathologies of the spinal cord. Progesterone, administered at the
onset of reperfusion, protected spinal cord neurons from ischemiainduced damage (Vandenberk et al., 2013). The studies performed on
animal models of multiple sclerosis, collectively referred to as experimental autoimmune encephalomyelitis (EAE), are particularly relevant
to ndings in SCI. Classic EAE is functionally characterized by ascending
paralysis. The histopathology of EAE includes spinal cord inammation,
demyelination and neuronal/axonal damage. In mice, progesterone
treatment, initiated prior to induction of EAE, attenuated the expression

of pro-inammatory mediators including TNF- and its receptor TNFR1


and reduced microglial reactivity in the spinal cord (De Nicola et al.,
2013a). These ndings, together with earlier reports showing neuroprotective and promyelinating effects of progesterone in EAE (Garay et al.,
2009, 2012) or in other pathological conditions of the CNS (for reviews
see Stein, 2011; Schumacher et al., 2007), support the idea that progesterone could improve the outcomes of spinal cord inammation and
injury. Interestingly, in the Wobbler mouse, spinal cord motor neuron
pathology is also alleviated by progesterone (De Nicola et al., 2013b).
Even so, it is important to keep in mind that investigations showed
that natural progesterone or synthetic progestins might differ in effects
and even exert opposing actions. For example, while some studies
reported benecial effects of MPA on lipid peroxidation (Topsakal
et al., 2002) or TNF- levels (Sahin et al., 2011) in the injured spinal
cord, others found that MPA antagonized the protective actions of estrogens against glutamate excitotoxicity in hippocampal neurons, in vitro
(Nilsen and Brinton, 2003). Yet, Nilsen and Brinton (2002) indicated
that progesterone and 19-norprogesterone, the progesterone agonist
which is used in clinics, alone or in combination with estrogen, exerted
protective effects against glutamate excitotoxicity.
Information about the role of testosterone in neuroprotection versus
neural damage in SCI is limited. Even in other pathological conditions of
the CNS, the benecial or detrimental role of testosterone is still debated. As already mentioned, testosterone can be aromatized to estradiol in
the CNS and some of its benecial effects could be mediated via estrogen
receptors rather than androgen receptors. As pointed out above, the
effects of testosterone on gliosis have been attributed to its aromatization into estradiol (Garcia-Estrada et al., 1993). In agreement with this
concept, the positive effects of testosterone on hippocampal synaptic
transmission during EAE are not observed when the non-aromatizable
androgen DHT is used (Ziehn et al., 2012). These studies raise the possibility that testosterone therapy could potentially substitute estrogen
therapy in men with SCI.
In summary, although pre-clinical investigations suggest that 17estradiol, ER agonists and progestogens hold promise as neuroprotective agents in SCI, it is important to emphasize that many issues need
to be further addressed in pre-clinical investigations before considering
sex steroids as therapeutic agents for neural protection in the clinical
setting. An opinion survey, about the level of preclinical evidence that
is required for translating potential neuroprotective/neuroregenerative
therapies into clinical trials in SCI, revealed that most researchers agreed

Table 2
Summary of SCI paradigms, progesterone or progestin treatment regimen and outcome measures used in rats.
SCI paradigm

Progesterone regimen
Dose/route

T8 contusion injury in male rats


T10 complete transection in male rats
T4T5 compression injury in male rats
T10 complete transection injury in
male rats
T10 complete transection in male rats

Major outcome measures


Time of administration

4 mg/kg; i.p.

30 min followed by 6, 24,


48, 72, 96 and 120 h p.i.
4 mg/kg, i.p. and s.c. 1 (i.p.) followed by 24,
48 and 72 h (s.c.) p.i.
60 mg/kg MPA, i.m. Immediately after SCI
4 mg/kg; i.p. and s.c. 1 h (i.p.) followed by 24,
48 and 72 h (s.c.) p.i.
4 mg/kg, i.p. and s.c. 1 h (i.p.) followed by 24,
48 and 72 h (s.c.) p.i.

BBB

Spared white matter

Thomas et al. (1999)

None

Properties and survival of ChAT positive


neurons in ventral horn
Markers of lipid peroxidation
BDNF expression, chromatolysis

Labombarda et al.
(2002)
Topsakal et al. (2002)
Gonzalez et al. (2005)

Survival of NG2+ oligodendrocyte


precursors, myelin basic protein
expression
Total spared tissue

Labombarda et al.
(2006)

None
None
None

T10 contusion injury in male and


female rats
T10 complete transection in male rats
T10 complete transection in male rats

4 mg/kg; 8 mg/kg
or 16 mg/kg, i.p.
4 mg/kg, s.c.
16 mg/kg/day, s.c.

30 min, 6 h, p.i. followed by daily BBB


injections for 5 or 14 days p.i.
1 h followed by 24, 48, 72 h p.i. None
Daily starting on day of SCI
None

T13 hemisection in male rats

16 mg/kg/day, s.c.

Daily starting on day of SCI

T10 complete transection in male rats

16 mg/kg/day, s.c.

Daily injections starting at


3 h p.i., for 3 or 21 days
Immediately after SCI

T10T12 compression injury in male rats 8 mg/kg MPA, i.p.

Reference

Functional tests Histological/molecular analyses

Von Frey hair


acetone-cold
allodynia
None
None

Fee et al. (2007)

Chromatolysis, MAP2 immunoreactivity


Myelin proteins, oligodendrocyte
differentiation
Molecular components of central pain
mechanisms

Gonzalez et al. (2009)


Labombarda et al.
(2009)
Coronel et al. (2011)

Gliosis, oligodendrocyte differentiation

Labombarda et al.
(2011)
Sahin et al. (2011)

TNF- and IL-1 levels

S. Elkabes, A.B. Nicot / Experimental Neurology 259 (2014) 2837

with the idea of demonstrating the effectiveness of non-invasive drug


therapy in large animal models of SCI before initiating human trials
(Kwon et al., 2010, 2012). When individuals with SCI responded to
the same query, the majority declared strong support for the evaluation
of drug efcacy in large animal models, even though a large number
also thought that showing drug effectiveness in rodent SCI models is
sufcient to justify clinical trials (Kwon et al., 2012). Preclinical investigations on large animal models of SCI could be helpful to further corroborate sex steroid-dependent neuroprotection before implementation
of costly, scarce and challenging clinical trials. In addition, delaying the
initiation of intervention to subacute/chronic phases and assessing the
long-term effects of sex steroids in clinically relevant, severe SCI models
are additional points to consider.
Acknowledgments
This work was supported by Rgion Pays de la Loire (AN), New
Jersey Commission on Spinal Cord Research (SE) and Reynolds Family
Spine Laboratory.
This review was envisioned as a concise overview of the sex steroids,
neuroprotection and SCI eld; we apologize to those who published
excellent reports in this area but were not cited here.
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