Neurosteroids and Ischemic Stroke: Progesterone a Promising Agent in Reducing the Brain Injury in Ischemic Stroke

Abstract

Progesterone (P4), a well-known neurosteroid, is produced by ovaries and placenta in females and by adrenal glands in both sexes. Progesterone is also synthesized by central nervous system (CNS) tissues to perform various vital neurological functions in the brain. Apart from performing crucial reproductive functions, it also plays a pivotal role in neurogenesis, regeneration, cognition, mood, inflammation, and myelination in the CNS. A substantial body of experimental evidence from animal models documents the neuroprotective role of P4 in various CNS injury models, including ischemic stroke. Extensive data have revealed that P4 elicits neuroprotection through multiple mechanisms and systems in an integrated manner to prevent neuronal and glial damage, thus reducing mortality and morbidity. Progesterone has been described as safe for use at the clinical level through different routes in several studies. Data regarding the neuroprotective role of P4 in ischemic stroke are of great interest due to their potential clinical implications. In this review, we succinctly discuss the biosynthesis of P4 and distribution of P4 receptors (PRs) in the brain. We summarize our work on the general mechanisms of P4 mediated via the modulation of different PR and neurotransmitters. Finally, we describe the neuroprotective mechanisms of P4 in ischemic stroke models and related clinical prospects.

Full text

Journal of Environmental Pathology, Toxicology and Oncology, 36(3):191–205 (2017)
191
0731-8898/17/$35.00 © 2017 Begell House, Inc. www.begellhouse.com
Neurosteroids and Ischemic Stroke: Progesterone
a Promising Agent in Reducing the Brain Injury in
Ischemic Stroke
Syed Suhail Andrabi, Suhel Parvez, & Heena Tabassum*
Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110062, India
*Address all correspondence to: Dr. Heena Tabassum, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard
University), New Delhi 110 062, India; Tel.: +91 11 26059688x5573; Fax: +91 11 26059663, E-mail: tabazsum9@gmail.com or
heenatabassum@jamiahamdard.ac.in
ABSTRACT: Progesterone (P4), a well-known neurosteroid, is produced by ovaries and placenta in females and by
adrenal glands in both sexes. Progesterone is also synthesized by central nervous system (CNS) tissues to perform various
vital neurological functions in the brain. Apart from performing crucial reproductive functions, it also plays a pivotal
role in neurogenesis, regeneration, cognition, mood, inammation, and myelination in the CNS. A substantial body of
experimental evidence from animal models documents the neuroprotective role of P4 in various CNS injury models,
including ischemic stroke. Extensive data have revealed that P4 elicits neuroprotection through multiple mechanisms
and systems in an integrated manner to prevent neuronal and glial damage, thus reducing mortality and morbidity.
Progesterone has been described as safe for use at the clinical level through dierent routes in several studies. Data
regarding the neuroprotective role of P4 in ischemic stroke are of great interest due to their potential clinical implications.
In this review, we succinctly discuss the biosynthesis of P4 and distribution of P4 receptors (PRs) in the brain. We
summarize our work on the general mechanisms of P4 mediated via the modulation of dierent PR and neurotransmitters.
Finally, we describe the neuroprotective mechanisms of P4 in ischemic stroke models and related clinical prospects.
KEY WORDS: progesterone, stroke, model, rat, mice, ischemia, neuroprotection
I. INTRODUCTION
Thousands of people are being aected by neuro-
logical diseases every year, with no hope of altering
the course of their disease with current therapeutic
options; thus, it is imperative to nd new therapeu-
tic avenues.1 Such neurological diseases include Al-
zheimer’s disease (AD), Parkinson’s disease (PD),
Huntington’s disease (HD), amyotrophic lateral
sclerosis (ALS), motor neuron degeneration (MND),
Multiple Sclerosis (MS), epilepsy, stroke, and lesser-
known frontotemporal dementia. Various etiological
factors (e.g., genetic, epigenetic, physiological, and
environmental factors) contribute to pathophysiology
of these neurological diseases, which pose a great
economic burden on these patients and their families.2
For example, stroke is a neurological disorder that af-
fects millions of people worldwide.3 Ischemic stroke
is a prevalent form of stroke with few eective thera-
peutic options that are useful at the clinical level.1 Due
to a lack of eective treatment that can alter course of
this disease, researchers are trying to nd new thera-
peutic strategies to improve clinical outcomes.2 Vari-
ous endogenous compounds, nutraceuticals, and syn-
thetic compounds have been implicated in dierent
neurological diseases (e.g., PD, AD, and HD) for their
neuroprotective properties; of these, neurosteroids
are the most promising candidates. Neurosteroids
are synthesized in nervous system and are involved
in several reproductive and nonreproductive events
like cognition, mood change, appetite, locomotory
activity, and nerve cell survival.3 Neurosteroids are
critical for neurodevelopment, neuronal activity, neu-
ronal proliferation, and dierentiation.4 Apart from
these functions, neurosteroids also help in regulating
respiration and mitochondrial membrane potential to
maintain mitochondrial homeostasis.4 Neurosteroids
also regulate a number of neurotransmitters including
GABA, glutamate, epinephrine, norepinephrine, ace-
tylcholine, dopamine and serotonin.5 Neuroprotective

Journal of Environmental Pathology, Toxicology and Oncology
Andrabi, Parvez, & Tabassum
192
mechanisms of neurosteroids regulate various apop-
totic pathways, signaling cascades, and neuronal
signaling.5 Many neuroactive steroids [e.g., estradi-
ol, estrone, pregnenolone, pregnenolone isosulfate,
allopregnenolone (ALLO), dehydroepiandrosterone
(DHEA) and P4] have neuroprotective properties.6
Among these neurosteroids, P4 has been shown by
various preclinical and clinical trials to be one of the
most promising agents for the treatment of various
diseases like prostate cancer, osteoporosis, diabetic
neuropathy, and neurological disorders.7 Although
P4 is an ovarian hormone, it is also produced by
various brain cells and performs several neurologi-
cal functions, making it one of the most important
neurosteroids of the CNS.8 In the CNS, it helps reg-
ulate a wide range of functions that are important
for normal physiological processes in maintaining
the homeostasis between CNS and the peripheral
nervous system (PNS).9 Progesterone functions as
a neuromodulator, neurogenic molecule in addition
to playing an important role in neurotransmission
and myelination.10 It also induces various develop-
mental processes through neurotrophic factors that
combine dendritic growth, spinogenesis, and syn-
aptogenesis in cerebral neurons. Other than these
physiological functions, P4 may serve as a thera-
peutic option for various psychiatric disorders.11 Its
anti-inammatory property has been utilized in neu-
roinammatory diseases like MS.12,13 After decades
of research, P4 has proven eective in various ani-
mal models of neurological diseases including trau-
matic brain injury (TBI), spinal cord injury (SCI),
MS, seizures, AD, PD, and stroke.14 These neuro-
protective properties of P4 indicate the opportunity
for further investigation in various animal models of
ischemic stroke for therapeutic alternatives that may
be helpful at the clinical level.15 In this review, we
describe dierent aspects of P4 and possibilities for
exploiting its neuroprotective property in ischemic
stroke.
II. PROGESTERONE: NEUROPROTECTIVE IN
NEUROLOGICAL DISEASES
Progesterone is one of the most attractive pharma-
cological agents among neurosteroids because it
is a multifunctional molecule having multiple tar-
gets.16 Its potential therapeutic applications have
been widely explored in a number of neurologi-
cal diseases including ischemic stroke.17 In TBI,
P4 has been able to salvage the traumatic injury in
a number of preclinical and clinical studies.18 The
wobbler mouse model is an important model for
neurodegenerative diseases such as ALS and spi-
nal muscular atrophy.18 Progesterone treatment in
wobbler mice attenuated the number of brain in-
juries and alleviated the motor neuronal degenera-
tion via dierent mechanisms including regulation
of mitochondria, brain-derived neurotrophic factor
(BDNF), and GABAergic neurons.18 Progesterone
reduces amyloid beta (Aβ)–induced pathological
conditions in rats. This nding might be useful in
future therapeutic strategies in AD.19 6-OHDA–in-
duced cognitive impairments and loss of learning
and memory in hemi-Parkinsonian rats have been
modulated by P4 treatment.20 Huntington’s disease
is a neurodegenerative genetic disorder that leads
to a decline in mental and behavioral functions;
these symptoms were attenuated by P4 administra-
tion in a 3-nitropropionic acid–induced HD model
in rats.21 Progesterone also attenuated neurode-
generation in a murine model of G93A-SOD1 of
ALS.22 A number of studies have shown a protec-
tive role of P4 in spinal cord injury through pro-
myelinating and anti-inammatory properties.23
Apart from these neurological diseases, P4 elicits
its neuroprotection in ischemic stroke via number
of mechanisms that are discussed further in this re-
view.
III. PROGESTERONE BIOSYNTHESIS AND
METABOLISM
The rst de novo neuronal synthesis of neuros-
teroids was the pioneering discovery by Baulieuand
et al.24 Neurosteroids are produced by nerve cells
through a process known as neurosteroidogenesis,
and they are regulated by various steroidogenic en-
zymes present in dierent regions of brain tissue.25
Progesterone is produced within the CNS and PNS
by neurons, astrocytes, and glial cells, thus quali-
fying it as a neurosteroid.26 Purkinje cells, the prin-

Volume 36, Issue 3, 2017
Progesterone as Neuroprotectant 193
ciple cerebral neurons, are considered an impor-
tant site for synthesis and metabolism of P4 in the
brain. These cerebellum neurons are considered
important models for the study of neurosteroid
metabolism.27 These Purkinje cells contain various
steroidogenic enzymes involved in synthesis of
P4: cytochrome P450, side-chain cleavage enzyme
(P450scc), and 3β-hydroxysteroid dehydrogenase/
Δ5-Δ4-isomerase (3β-HSD).28 However, to initiate
this process, nerve cells need a primary cholesterol
product that is de novo synthesized in brain cells.29
In the brain, P4 is synthesized from de novo cho-
lesterol and pregnenolone, which is either derived
from circulation or cleavage of a cholesterol side
chain by cytochrome P450scc.29 Cholesterol must
be transported to the inner mitochondrial mem-
brane from intracellular stores to initiate the syn-
thesis of P4.30 This rate-limiting step in pregneno-
lone formation is regulated by two mitochondrial
membrane proteins: peripheral benzodiazepine re-
ceptor (PBR) and steroidogenic acute regulatory
protein (StAR).31 These proteins are responsible
for intramitochondrial transport of cholesterol and
are widely expressed in brain cells.32 To initiate
P4 formation, the cholesterol side-chain ring is
cleaved by an enzyme P450scc, yielding pregnen-
olone.33 Pregnenolone formed in the mitochondria
is then transported into microsomal compartments
where it is converted into P4 by 3β-HSD.33 After
the formation of P4 in the neurons, astrocytes, and
glial cells, it is metabolized into various neuroac-
tive compounds such as 5α-dihydroprogesterone,
ALLO, and androstenedione. Progesterone is
metabolized into 5α-dihydroprogesterone by
5α-reductase and ALLO by another steroid enzyme,
3α-hydroxysteroidoxidoreductase (3α-HOR).34
Progesterone is also metabolized into androstene-
dione by another enzyme, P45017α, lyase which is
then converted into number of steroids (e.g., tes-
tosterone, estrone, and estradiol-17β) by dierent
enzymes.35 These metabolites of P4 play pivotal
roles in physiological and pathophysiological con-
ditions.36 Studies have shown that ALLO, one of
these important metabolites, has also been shown
to function as a neuroprotectant in a number of
brain injury models (Fig. 1).37
IV. PROGESTERONE RECEPTORS IN THE
BRAIN
Apart from reproductive functions, P4 has vari-
ous CNS functions: regulation of cognition mood,
mitochondrial functions, inammation, neurogen-
esis, regeneration, myelination, and neuroprotec-
tion in SCI and TBI.38–41 Some of these functions
performed by P4 are mediated through dierent
receptors (i.e., isoforms) that are present through-
out the CNS and are widely expressed by brain
cells.42 Various types of receptors like nuclear and
membrane receptors are involved in the execution
of multiple functions of P4.43 These include clas-
sical nuclear P4 receptors (cPR), which are pres-
ent in dierent regions of CNS including the ce-
rebral cortex, cerebellum, the hippocampus, and
cortical structures.44 Two of these major receptor
isoforms, PRA and PRB, are encoded by the same
gene having eight exons.45 Progesterone receptor
B is more diverse in function than PRA because
of its larger size, with a 164 amino-acid sequence
in the N- terminal region.46 Various experiments
have shown another putative membrane protein,
Dx-25, through which P4 binds and imparts neu-
roprotection.47–51 It was cloned rst in the porcine
liver; later, a homologous protein was identied
in the rat (25-Dx), in mice (PGRMC1), and in hu-
mans (Hpr.6).52 Dx-25 is localized in several brain
regions: the hypothalamus, the circumventricular
area and ependymal cells, and the meninges.53 Dif-
ferent techniques (e.g., immunohistochemistry, u-
orescence immunolabelling, and confocal micros-
copy) have revealed that PGRMC1 is coexpressed
with a vasopressor by neurons of paraventricular
and supraoptic nuclei.5 The putative membrane re-
ceptor PGRMC1 is expressed in the Purkinje cell,
a cerebellar neuron.53 This membrane protein has
also been detected in the cerebellum of the rat by
RT-PCR and western blot analysis.54 Apart from
these nuclear receptors and a single P4 membrane
binding component (PGRMC1), other membrane
receptors, like mPR subtypes (mPRα, mPRβ and
mPRγ) through which P4 exerts neuroprotection,
have been found in various tissues, including the
brain.55–57 Recent studies have shown two other

Journal of Environmental Pathology, Toxicology and Oncology
Andrabi, Parvez, & Tabassum
194
types of mPR (mPRɛ and PRδ) localized in the
brain and spinal cord.50 All ve of these mPRs are
found in the brain and spinal cord, but their expres-
sion levels dier in dierent regions of the CNS.
mPRα is expressed and in mouse and rat models
of induced brain injury and may be responsible for
number of functions including neuroprotection,
cognition, and behavior.58 The σ-1 receptor present
in various organs, including the brain, is immersed
in lipid rafts of endoplasmic reticulum where it is
associated with mitochondria and has been studied
in number of neurological diseases.59 Progesterone
binding to the σ-1 receptor is involved in the mod-
ulation of various functions of this receptor, such
as ion-channel activity, which may be a part of its
neuroprotective mechanism.59
V. MECHANISM OF ACTION OF
PROGESTERONE
Progesterone modulates dierent physiological
processes through multiple mechanisms executed
in cumulative manner to promote hormone-depen-
dent actions. Mechanistically, P4 acts through ge-
nomic and nongenomic modulation of neurotrans-
mitters in the CNS,60,61 through processes executed
via dierent nuclear and membrane receptors.62
Progesterone, like other steroids, regulates its own
action by regulating the transcription of many
genes through interactions with many nuclear
and/intracellular receptors.63,64 The most common
PRs responsible for the transcription of genes are
PRA and PRB containing DNA-binding domain
and activation function 1 (AF-1) domain.43 These
PRs are found in a nonligated form in both the
cytoplasm and nucleus.65 Normally, a nonligated
form of PR found in cytoplasm is inactive and is
bound to suppressor heat-shock proteins.65 Once
P4 binds to PRs, they dissociate from the heat-
shock proteins and are translocated into nucleus,
where they bind to target genes through specic
palindromic response elements (PREs) for tran-
scription by recruiting transcription machinery.
They then perform dierent functions such as neu-
rogenesis and repair.62 This transcription-mediated
eect of P4 is a delayed one. However, P4 can also
prompt its action through another nonclassical and
nongenomic mechanism by binding with various
membrane receptors.63 Progesterone binds to vari-
ous membrane receptors and activates signaling
cascades through modulation of ion channels and
secondary messengers64,65 it binds with PGRMC1
and mPRs to activate various intracellular sig-
naling pathways that regulate dierent cell func-
tions.65 When P4 binds to PGRMC1, it activates
extracellular signal-related kinases (ERK).66 As a
part of its multiple mechanism cascades, the P4
metabolite ALLO acts as a positive modulator of
GABA receptor (GABAR).67 ALLO binds to hy-
drophobic domain of GABAR, which leads to its
potentiation by increasing the opening of GABA-
gated chloride channel.68 ALLO has been shown to
exert neuroprotection through GABAA-receptors
in hippocampal slices of rat.69 In another mecha-
nism, P4 directly regulates GABA by activating
signaling pathways, which leads to the phosphory-
lation of discrete sites of GABAR.70 The P4 me-
tabolite ALLO has been proven to be protective
in movement disorder via positive modulation of
GABAR in neuroleptic-induced dyskinesia.71–73
In another study, P4 and its metabolite mediated
neuroprotection through the mitochondria by in-
hibiting mitochondrial permeability transition pore
(mPTP). Furthermore, the inhibition of mPTP may
inhibit the release of apoptotic factors especially
cytochrome c (Cytc).74 The sigma-1 receptor is a
P4 binding receptor through which P4 elicits its
protective eect; it is involved in various cellular
functions such as the regulation of cellular cal-
cium level, oxidative stress, apoptotic pathways,
cell survival, and mitochondrial functions.75 Pro-
gesterone blocks the receptor that modulates ion
channel activity, especially the calcium channel,
and this function may be useful in future therapies
in ischemic stroke.76 The σ-1 receptor is involved
in neuroprotection in various brain injuries such as
TBI and ischemic injury.76 A P4 antagonistic eect
has been shown to reduce the intracellular calcium
level through the NMDA receptor, which salvages
the ischemic injury (Fig. 2).77

Volume 36, Issue 3, 2017
Progesterone as Neuroprotectant 195
VI. NEUROPROTECTIVE MECHANISM OF
PROGESTERONE IN ISCHEMIC STROKE
Much experimental evidence indicates that P4 has a
neuroprotective role in various models of CNS dis-
orders, including ischemic stroke (Table 1).78 Over
the last two decades, intense research has contin-
ued to elucidate the multiple mechanisms of P4 in
reducing ischemic injury in brain.79 Thus, data are
now available that yield mechanistic insight into
neuroprotective properties of P4 in ischemic stroke.
In 1996, a group of researchers found that P4 could
alleviate ischemic injury in a rat model of cerebral
ischemia by reducing the infarction. Progesterone
was given at the dose of 4 mg/kg body weight im-
mediately prior to middle cerebral artery occlusion
(MCAO) or 2 hours after reperfusion. Recovery
from ischemic injury was shown in P4-adminis-
tered animals, as indicated by better neurological
outcomes and less infarction.80–82 Successive studies
on P4 not only authenticated these ndings but also
produced new therapeutic avenues that will be help-
ful in designing the future pharmacological thera-
pies. The huge existing data set reveals a number of
mechanisms (e.g., antiapoptotic, anti-inammatory,
antioxidative, and modulation of number of signal-
ing pathways) through which P4 exerts neuropro-
tection in ischemic stroke.
VII. PROGESTERONE IMPROVES
NEUROLOGICAL DEFICITS AND
REDUCES INFARCTION INDUCED BY
ISCHEMIC STROKE
Neurological impairment is a hallmark of ischemic
Cholesterol
Pregnenolone
Progesterone Dihydroprogesterone Allopregnanolone
Androstenedione Estrone
Estradiol
Testosterone
Fig.1
FIG. 1: Schematic representation of progesterone (P4) synthesis and metabolism in brain cells. In the mitochon-
dria, the enzyme P450 side-change cleavage (P450scc) converts cholesterol into pregnenolone (PREG), which is
further transformed into P4 in the endoplasmic reticulum in neurons and astrocytes. In neurons, as well as in astro-
cytes, P4 and testosterone may be metabolized by the enzymatic complex formed by (5ɑ-R) and 3ɑ-hydroxysteroid
dehydrogenase (3ɑ-HSD) into dihydroprogesterone (DHP) and the tetrahydroprogesterone (THP), also known as
allpregnanolone. P4 is converted into androstenedione by P450c17 in neurons and astrocytes which further metabo-
lized into testosterone by 17ß-HSD. In neurons, androstenedione, as well as testosterone, may be converted by the
enzyme P450 aromatase (ARO) to estrone and 17b-estradiol (17b-E2), respectively. This occurs also in astrocytes,
which display ARO, but not in microglia. Arrows represents cellular localization in neurons, lines represent cel-
lular localization in astrocytes, and dots represent cellular localization microglia.

Journal of Environmental Pathology, Toxicology and Oncology
Andrabi, Parvez, & Tabassum
196
stroke, which leaves millions of people debilitated.
Studies have conrmed that P4 improves a number
of neurological decits in various neurological dis-
eases: motor impairment, somatosensory neglect,
locomotory activity, spatial navigation, learning,
and memory.83 In one study, investigators reported
that P4 signicantly improved the neurological
function decit in ovariectomized mice, while no
recovery was found in aged mice. Notably, P4 re-
duced the infarction volume in aged mice but not
in ovariectomized mice.84 Progesterone has also
been found to be neuroprotective in cerebral isch-
emia in Sprague-Dawley rats in an MCAO model,
as shown by improved rota-rod grip strength and
sensory neglect.85 Administration of P4 through
minipumps has been eective in eliciting neuro-
protection in adult mice (but not in aged mice) at
a dose regimen of 8 mg/kg body weight followed
by subcutaneous infusion of P4 at 1.0 µL/hour for
three consecutive days. The dose paradigm of 8
mg/kg has shown signicantly better neurologi-
cal outcomes in hypertensive mice compared with
hypertensive control mice.85 Delayed treatment of
P4 has been eective in improving behavioral de-
cits such as grip strength, motor coordination, spa-
tial navigation, learning, and memory at the dose
regimens of 8 and 16 mg/kg in permanent MCAO.
This suggests that 8 mg/kg could represent the
optimal dose over 16 and 32 mg/kg doses.86 In a
study designed to optimize the dose-response re-
lationship to determine the best dose paradigm,
8 mg/kg had better ecacy than 16 or 32 mg/kg.
Also, 16 mg/kg was more eective than 32 mg/
kg in improving neurological functions. At a dose
of 32 mg/kg body weight given after induction of
ischemia, there was no signicant reduction in in-
farction volume in an MCAO model of rats.86 Also,
P4 has been eective in the pMCAO model at the
dose of 8 mg/kg in Sprague-Dawley rats given at 1
hour (i.p.) followed by subcutaneous at 6, 24, and
48 hours after induction of ischemia, respectively.
Progesterone reduced the infarction volume and
improved neurological functional decits.87 In an-
other study on aged rats with pMCAO, P4 exerted
neuroprotection by decreasing infarction and im-
proving neurological decits.87
VIII. THE ANTIOXIDATIVE MECHANISM OF
P4 IN ISCHEMIC STROKE
Oxidative stress is one of the complex cascading
pathways involved in the pathophysiology of brain
injury in cerebral ischemia.88 Oxidative free radi-
cals produced during ischemia activate other apop-
totic and inammatory pathways that worsen the
brain injury.89 Antioxidants are the innate defense
system against the oxidative stress that damages
the macromolecules and leads to a number of neu-
rodegenerative diseases.90 Due to its antioxidative
property, P4 has been able to elevate the levels of
antioxidative enzymes (e.g., GPx, SOD, and cata-
lase) in an ischemic model of bilateral common ca-
rotid artery occlusion (BCAO). Progesterone also
alleviates the membrane damage in ischemic rats
by abrogating lipid peroxidation.91
IX. THE ANTIAPOPTOTIC MECHANISM OF
P4 IN ISCHEMIC STROKE
Mitochondrial dysfunction plays a critical role in
ischemic stroke via ROS generation and apoptosis.92
Apoptotic pathways are critical pathways that are
aected during ischemic brain injury; the present
a highly interesting target for potential therapeutic
strategies.93 Mitochondrial dysfunction induced by
ischemic factors leads to the formation of perme-
ability transition pores through which the apoptotic
protein cyt c translocates into cytoplasm and causes
neuronal death.94 Progesterone and its metabolite
ALLO impart neuroprotection through an antiapop-
totic property by inhibiting the mPTP, which in turn
inhibits the release of apoptotic factors such as cyt
c.74 One of the important antiapoptotic pathways,
PI3K/Akt, is upregulated by P4 and rescues the in-
jury-induced neuronal death. Progesterone has been
able to attenuate apoptosis through activation of
the PI3K/Akt pathway, which increases the expres-
sion of the antiapoptotic protein bcl-2 and decreases
the level of the apoptotic protein bax.95 One of the
important apoptotic proteins, Caspase-3, which ex-
ecutes the apoptotic pathway, is one of the targets of
P4 that leads to recovery of ischemic injury.96

Volume 36, Issue 3, 2017
Progesterone as Neuroprotectant 197
X. THE ANTI -INFLAMMATORY MECHANISM
OF PROGESTERONE IN ISCHEMIC
STROKE
Ample evidence indicates that inammation is a
double-edged sword; it exacerbates the second-
ary brain injury and contributes to brain recovery
after stroke. Currently, the cascades of inamma-
tory events are being explored for neuroprotective
therapies in ischemic stroke.97 Inammatory cas-
cades that potentiate the brain injury involve the
activation of microglia that release proinamma-
tory cytokines and other molecules [e.g., COX-2
(cycloxygenase-2), NF-kB, and iNOS], which are
modulated by P4 and may act as protectants in neu-
roinammation.97 Due to these anti-inammatory
properties, P4 inhibits the expression level of COX-
2 and ionized calcium-binding adapter molecule 1
(Iba1), providing neuroprotection in a pMCAO-
induced stroke model in rats.98 Progesterone’s neu-
roprotection as mediated through the attenuation
of COX-2 and IL-1β expression levels at a dose of
8 mg/kg body weight in an HIBD model of Wistar
rats.98 Progesterone has been able to exert neuro-
FIG. 2: Schematic diagram of mechanism of progesterone in brain that is supposed to lead neuroprotection in
ischemic stroke. Ischemic stroke involves various events such as oxidative stress, inammation, and excitoxicity,
which lead mitochondrial dysfunction. Progesterone of peripheral origin comes through blood circulation and local
progesterone binds to various receptors, such as mPR, PGRCM1, sigma-1, and nPR, to elicit its neuroprotective
eect. P4 binds to nPR and regulate the gene expression of various neurotrophic factors like BDNF, NGF, and
VEGF, which are involved in neuroprotection. BDNF is an important growth factor that binds TRKb receptors
and activates the signaling pathways such as PI3K/AKT pathway, MAPK pathway. These upregulate the level of
antiapoptotic protein (BCL-2) and attenuate the apoptotic protein (Bax, Bad), which directly inhibits the release
of cytochrome c into cytosol and inhibits neuronal death. P4 also inhibits the formation of mPTP, which stops the
release of death factors. P4 reduces oxidative stress by elevating the levels of GPX and catalase and decreases lipid
peroxidation, which attenuates ischemic injury. The P4 metabolite allpregnanolone binds to GABAR, which is
neuroprotective in ischemic stroke by inhibiting Ca2+.
Pregnenolone Progesterone5α-Dihyroprogeste rone Allopregnenolone
mPR PGRMC1 Sigma-1
P4P4 P4
P4
nPR
Geneexpression
GABAR
Bloodcirculation
BDNF
NGF
VEGF
TrkB
PI3K/AKT,MAPK,ERK1/2
…
Bcl-2
Bad
Bax
Cytochromec
Inflammation
IL-1,IL-6,TN F-α,NF-κB
OxidativeDamage Mitochondrialdysfunction
Apoptosis
Necrosis
LPO
GPx Cat
Ischemicinjury
P4
SOD
P4 P4
Fig.2

Journal of Environmental Pathology, Toxicology and Oncology
Andrabi, Parvez, & Tabassum
198
protection by alleviating the inammation-induced
intercellular adhesion molecule-1 (ICAM-1), vas-
cular cell adhesion molecule-1 (VCAM-1). In this
study, P4 also attenuated the expression of macro-
phage marker CD68 and myeloperoxidase activity
(MPO) in rats with pMCAO.98 In an another study
TABLE 1: Neuroprotective mechanisms mediated by progesterone in rodent models of ischemic
stroke
Model Dose Route/Duration Probable Mechanism Ref.
Rat 8 mg/kg b.w. Postocclusion, i.p., 1 h followed
by s.c. injection at 6, 24, and
48 h
MCP-1 and CXCL-1↓107
Rat 8 mg/kg b.w. 30 min before occlusion, i.p. COX-2 and IL-1β↓89
Rat 8 mg/kg b.w. After reperfusion, i.v., at 15 min,
2, 6, 24, 48, and 72 h
caspase-3, DNA fragmenta-
tion
100
Rat 15 mg/kg b.w. At 1 and 6 h postocclusion, i.p. Iba-1 and COX-2↓103
Rat 8 and 16 mg/
kg b.w.
After reperfusion, 2 h, i.p., 6 h,
every 24 h for 7 days, s.c.
IL-6, IL-1β, and TNF-α↓,
BDNF ↑
123
Rat 8 mg/kg b.w. Postocclusion at 1 h, i.p., fol-
lowed by s.c. at 6, 24, and 48 h
pAKT/anti-akt ↑;
BAD, pBAD, caspase-3, and
VEGF↓; BDNF↑
111
Rat 8 mg/kg b.w. 1 h postocclusion followed by
every day for 6 days, s.c.
ICAM-1, VCAM-1,CD68,
MPO, and TTC ↓
104
Rat 8 mg/kg b.w. 5 min, i.p., prior to reperfusion BDNF/TrkB/Erk1/2 ↑; IL-6
and NF-ᶄB ↓; BCl-2 ↑;
cleaved caspase-3 ↓
111
Rat 8 mg/kg b.w. 2 h postocclusion, i.p., then s.c.
at 6 h
MMP-9 and VEGF↓113
Rat 8 mg/kg b.w. i.p., 3, 6, or 24 h post s.c.; 5 h
later and then at every occlusion
GFAP, VEGF, and MMP9 ↓111
Rat 8 mg/kg b.w. Postocclusion, i.v., at 15 min, 2,
4, 6, 24, 48, and 72 h
Nogo-A, Ng-R, and Rho-A
↓
111
Rat 8 mg/kg b.w. Preocclusion, i.p., 30 min
MCAO
TNF-α and NF-κB ↓110
Rat 8 mg/kg b.w. Preocclusion, i.p., 30 min GSK-3β ↓; pAkt ↑110
Rat 4 mg/ kg b.w. pMCAO, i.p., at 6, 24, and 48 h PI3K, Akt, GSK3, and
β-catenin ↑
100
Mouse 8 mg/kg b.w. At the time of occlusion fol-
lowed by 6 h injection, i.p.
iNOS ↓101
Mouse 8 mg/kg b.w. 1, 6, and 24 h post MCAO, i.p.TGF-β2, NOS-2, and IL-1β
↓
105
Mouse 15 mg/kg/
b.w./day
30 min before ischemia, fol-
lowed by 24, 48, and 72 h
postocclusion, i.p.
TNF-α, LPO ↓;
GPx, SOD, and catalase↑
95
b.w., body weight; i.p., intraperitoneal injection; s.c., subcutaneous injection; i.v., intravenous injection; ↑, in-
crease; ↓ decrease.

Volume 36, Issue 3, 2017
Progesterone as Neuroprotectant 199
by the same group, P4 and its metabolite ALLO
showed neuroprotection in a pMCAO model at
a dose of 8 mg/kg 1 hour after induction of isch-
emia followed by subcutaneous doses at 6, 24, and
48 h post induction. Progesterone and ALLO de-
creased the levels of metalloproteinases (MMPs),
interleukin 6 (IL-6), and blood brain barrier (BBB)
disruption. Progesterone and ALLO signicantly
elevated the levels of junction proteins occulu-
din-1 and claudin-5 compared to the pMCAO con-
trol group.99 In a stroke model of HIBD at a dose
of 8 mg/kg, P4 reduced the expression of TNF-α
and NF-ĸB in hippocampal neurons. In this study,
P4 reduced the neuronal cavitations in the hippo-
campus.100 The anti-inammatory properties of P4
have been exploited for tPA-induced inammation
in a stroke model of rats. One study revealed that
P4 exerts its anti-inammatory property by modu-
lating microglia/macrophages factors that could
exacerbate the ischemic injury.101 In a recent study,
investigators showed that P4 preserved vascular-
ization at a dose level of 8 mg/kg/day in Sprague-
Dawley rats that had undergone transient middle
cerebral artery occlusion (tMCAO). This study
suggested that P4 inhibits the macrophage inltra-
tion in endothelial cells by reducing the chemotac-
tic protein [e.g., chemokine ligand-1(CXCL1) and
monocyte chemoattractant protein-1 (MCP-1)] in
ischemic endothelial cells that may salvage the
ischemic injury.102
XI. PROGESTERONE NEUROPROTECTION
THROUGH MODULATION OF VARIOUS
SIGNALING PATHWAYS
The nongenomic mechanism is a prompt-acting
pathway that involves the modulation of various
signaling pathways and leads to neuroprotection.
Various pathways are involved in ischemic stroke
such as pAkt/Akt, ERK, apoptotic, and inam-
matory pathways.103 The role of phosphoinositide
3-kinase/protein kinase B (PI3K/Akt) has been
implicated in several signaling pathways in-
volved in cell growth, cell survival, metabolism,
and inammation. Various studies have shown
that after ischemia, levels of pAKt increase; this
eect is believed to provide the neuroprotective
role in ischemic stroke.104 Progesterone activates
the P13K/Akt pathway, which salvages ischemic
injury through modulation of neurotrophic factors
like brain-derived neurotrophic factor (BDNF)
and attenuation of vascular epidermal growth
factor (VEGF) in pMCAO.105 Brain-derived neu-
rotrophic factor is an important neurotrophic fac-
tor responsible for neuroprotection in cerebral
ischemia, and P4 has been able to elevate the ex-
pression of mature BDNF.106 Vascular epidermal
growth factor is an important regulator of angio-
genesis and is implicated in ischemic brain in-
jury.107 Progesterone has been shown to alleviate
the levels of VEGF-MMP pathway, leading lead
to neuroprotection in ischemia.108 The TrKB re-
ceptor is one of the crucial neurotrophic receptors
involved in cell dierentiation and cell survival;
it is also involved in neuroprotection in ischemic
stroke via BDNF. BDNF binds to a TrKB recep-
tor that activates number of pathways (e.g., PI3K/
AKT, MAPK, and ERK1/2), which increases the
Bcl-2 protein level and leads to inhibition of cyt
c release into cytosol.109,110 Progesterone has been
shown to elicit neuroprotection through TrKB-
BDNF by increasing the gene expression of BDNF
through nPR.111 Glycogen synthase kinase-3β
(GSK-3β) has a proapoptotic function in hypoxic
ischemia by activating p53, which leads to apop-
tosis. Progesterone exerts its protective eect on
ischemic brain injury by inhibiting the expression
of GSK-3, which decreases the apoptosis through
the PI3K/Akt/GSK-3β pathway. This mechanism
has potential for use in developing drugs for treat-
ing ischemic brain injury.112
XII. CONFLICTING STUDIES
A few conicting reports contrast with results
showing the neuroprotection function of P4 in
ischemic injury. Murphy et al. reported that a
chronic dose of P4 before induction of ischemic
injury for 7 to 10 days augmented the ischemic in-
jury at the dose regimen of 30 or 60 mg/kg body
weight. Some of these interesting ndings suggest
that P4 could not attenuate ischemic injury if it

Journal of Environmental Pathology, Toxicology and Oncology
Andrabi, Parvez, & Tabassum
200
was administrated over a range of physiological
doses. One study showed that chronic use of P4
exacerbated the infarction in ovariectomized fe-
male rats.112 In another contradictory study, dose
played an important role in eliciting the eect of
P4. As previously described, 8 mg/kg has been
determined to be one of the most eective doses
to attenuate ischemic injury. These investigators
reported that 16 or 32 mg/kg could not elicit any
protection and that there had been no improvement
in neurological functions at those higher doses.113
Treatment with P4 before occlusion for 21 days
in ovariectomized female mice could not salvage
the ischemic injury, and no improvement in neuro-
logical functions and no modulation of ipsilateral
aquaporin-4 (AQP-4) expression were observed.
In addition, hormone treatment could not reduce
the lesion volume; thus, the researchers concluded
that prior treatment was not benecial for attenu-
ating ischemic injury.114 In postoperative tempera-
ture conditions, P4 and ALLO did not produce any
neuroprotection in ischemic hypertensive rats. The
ndings of this study contradict previous ndings
in which temperature and hypertension were not
considered. Thus, these agents might not show
any neuroprotection at normal body temperature.
Considering these contradictory studies and a mul-
titude of corroborative studies, we conclude that
dose, duration, and timing of hormone treatment
and maintenance of temperature are among the de-
cisive factors in eliciting the eect neuroprotective
eects of progesterone.
XIII. CLINICAL PROSPECTS OF
PROGESTERONE IN ISCHEMIC STROKE
Despite the enormous amount of preclinical data
available, some gaps remain to be overcome be-
fore these ndings can be translated at a clinical
level. The inconsistency in preclinical studies and
various unsuccessful clinical trials prompted the
Stroke Therapy Academy Industry Roundtable
(STAIR) to frame guidelines for preclinical test-
ing of candidate drugs.115 STAIR recommends
the use of more clinically relevant models with
greater reproducibility and has also called for a
dose-response study to evaluate the ecacy of
candidate drugs.116 It is imperative to determine
the therapeutic window of P4 administration us-
ing a pMCAO model that mimics the stroke in-
jury caused in humans; this is also recommended
by STAIR. Progesterone has been shown to be
safe at dierent dose regimens through dierent
routes at the clinical level.115 Two phase II clinical
trials show that P4 produces positive outcomes in
acute traumatic brain injury (TBI) patients.115 De-
spite the promising outcomes of phase II trials,
unfortunately, no satisfactory results were pro-
duced in a phase III trial by the National Institutes
of Health (NIH), which led to the termination of
the PROTECT III trial.117 However, the failure of
the phase III clinical trial might have been due
to incorrect extrapolation of preclinical data and/
or lack of systematic clinical trials, such as lim-
ited assessment of TBI.118 Novel comprehensive
strategies need to be designed at the clinical level
to translate the encouraging preclinical data suc-
cessfully from bench to bedside. These eorts
should include patient proling, dose paradigms,
timing of dose, and use of targeted outcome.
Considering that stroke and TBI culminate in the
same outcome, these ndings can provide a basis
for future clinical trials of P4 in stroke patients.
Successful phase III trials of P4 in acute TBI pa-
tients could be a big leap for brain injury therapy,
including stroke therapy.119–123
XIV. CONCLUSION AND FUTURE
PERSPECTIVES
Large and rapidly growing preclinical evidence has
shown that P4 is neuroprotective in various CNS
injury models like SCI, TBI, and ischemic stroke
injury. Progesterone has an advantage because it
elicits neuroprotection through multiple mecha-
nisms. These include antiapoptotic, antioxidative,
anti-inammatory mechanisms, and modulation of
various signaling cascades involved in ischemic
stroke. In two phase II clinical trials, P4 has been
eective in reducing the injury in acute TBI pa-
tients. Despite this large volume of preclinical data
and positive phase II clinical outcomes in TBI pa-

Volume 36, Issue 3, 2017
Progesterone as Neuroprotectant 201
tients, P4 has failed to show any positive outcome
at the phase III trial level. Several research gaps
may be responsible for the negative outcomes in
TBI patients, which have prevented P4 from being
translated into candidate drug for these patients.
Nevertheless, P4 may yet be eective for treat-
ments in other brain injury models like ischemic
stroke. At the preclinical level in ischemic stroke,
P4 has been eective in reducing brain cell dam-
age, but these ndings are, as yet, insucient to
bring P4 to application at the clinical stage. Further
preclinical studies are needed to evaluate the thera-
peutic window and dose-response of P4 in animal
models that will simulate the clinically relevant
models of ischemic stroke. These parametric data
are needed to support an investigational new drug
application before it can be tested at the clinical
level for ischemic stroke.
ACKNOWLEDGMENTS
SSA is a recipient of a Senior Research Fellow-
ship from University Grants Commission-Basic
Science Research [(UGC-BSR), Grant No.-
F-7/91/2007). The Grant no. [SR/PURSE Phase
2/39 (G)], received as PURSE grant from Depart-
ment of Science and Technology, Government of
India is thankfully acknowledged.
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