Future Directions in the Treatment of Neuropathic Pain: A Review on Various Therapeutic Targets

Abstract

Neuropathic pain is caused by structural lesion leading to functional abnormalities in central and peripheral nervous system. Neuropathic pain in itself is not always a disease, as it arises due to consequences of other diseases like diabetes, spinal cord injury, degenerative neuronal diseases and cancer. Current strategies of neuropathic pain treatment have provided relief to the patients to some extent, but complete cure is still a distant dream. In the future, it is hoped that a combination of new and improved pharmaceutical developments combined with careful clinical trials and increased understanding of neuroplasticity will lead to improved and effective pain management strategies leading to improved quality of life. In this review we have discussed about the various therapeutic targets of neuropathic pain and their pathophysiological mechanisms. Current status of drugs used for treatment of neuropathic pain have also been discussed in the review.

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CNS & Neurological Disorders - Drug Targets, 2014, 13, 63-81 63
Future Directions in the Treatment of Neuropathic Pain: A Review on
Various Therapeutic Targets
Matharasala Gangadhar, Ram Kumar Mishra, Dharmarajan Sriram and Perumal Yogeeswari*
Neuropathic Pain Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani,
Hyderabad Campus, R.R. District-500078, Andhra Pradesh, India
Abstract: Neuropathic pain is caused by structural lesion leading to functional abnormalities in central and peripheral
nervous system. Neuropathic pain in itself is not always a disease, as it arises due to consequences of other diseases like
diabetes, spinal cord injury, degenerative neuronal diseases and cancer. Current strategies of neuropathic pain treatment
have provided relief to the patients to some extent, but complete cure is still a distant dream. In the future, it is hoped that
a combination of new and improved pharmaceutical developments combined with careful clinical trials and increased
understanding of neuroplasticity will lead to improved and effective pain management strategies leading to improved
quality of life. In this review we have discussed various therapeutic targets of neuropathic pain and their
pathophysiological mechanisms. Current status of drugs used for treatment of neuropathic pain have also been discussed
in the review.
Keywords: Antidepressants, ion channels, microglia, neuropathic pain, neurotrophins, protein kinase M zeta, rho kinase.
INTRODUCTION
Pain is an unpleasant subjective perception usually in the
context of tissue damage. According to the International
Association for the Study of Pain (IASP) [1], pain is defined
as an unpleasant sensory and emotional experience
associated with actual or potential tissue damage or
described in terms of such damage. Pain, in general is a
natural protective mechanism for an organism as it alerts the
individual against harmful elements of the environment (like
heat, cut, pressure etc.). This pain is indicative and is known
as pathological pain. Inflammatory pain is reversible pain
produced due to tissue damage leading to inflammatory
response. It elicits physiological response to promote
healing. Pain is sensed and transmitted to the central nervous
system (CNS) by dorsal root ganglia (DRG). A DRG neuron
has three parts (a) pain sensing nociceptor terminal (b) axon,
which conducts the nociceptive signal and (c) pre-synaptic
terminal, which transmits the signal to the next neuron.
Likewise the signal is transmitted to the brain and is
interpreted as pain.
Depending on severity, pain can be divided as acute pain
and chronic pain. Acute pain usually starts suddenly and has
limited duration, whereas chronic pain lasts longer than
acute pain and is generally associated with long term illness.
On the basis of tissue type, pain can be further divided in
two categories: neuropathic pain and nociceptive pain.
Neuropathic pain, also called as nerve pain is primarily
caused by dysfunction in nervous system. Depending on the
site, it could be further divided in central and peripheral
neuropathic pain (see Fig. 1). The former is mainly caused
due to damage to spinal cord or brain, whereas later is
*Address correspondence to this author at the Birla Institute of Technology &
Science-Pilani, Hyderabad Campus. R.R. District-500078, Andhra Pradesh,
India; Tel: +91-9705932091; Fax: +91-40-66303998;
E-mail: pyogee@hyderabad.bits-pilani.ac.in
caused due to disease condition. Unlike physiological pain,
neuropathic pain doesn’t protect the body; rather it’s a
disease in itself. From a behavioral viewpoint, pathological
and inflammatory pain can be considered as adaptive tool for
better survival whereas neuropathic pain as maladaptive [2].
Neuropathic pain is a persistent type of pain and not only it
deteriorates the overall quality of life of the patient but it
also adds burden in terms of medical expenses.
Fig. (1). Classification of pain types.
In order to enable scientists to understand the
pathophysiological and molecular mechanisms, various
animal models have been used in past. Since neuropathic
pain as a whole, is a disease in itself or can be obtained as a
consequence of other diseases, scientists have relied on
animal models, specially rodent models. These models can
be divided in two categories: (a) nerve injury models: A
particular nerve is surgically injured to induce pain in animal
and (b) models mimicking a particular disease condition (see
Table 1).
Pain
Acute Chronic
Nociceptive
Pain
Neuropathic
Pain
Central
Peripheral
1996-3181/14 $58.00+.00 © 2014 Bentham Science Publishers

64 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 Gangadhar et al.
Till date, various mediators have been identified that are
involved in pain mechanisms. Tumor necrosis factor alpha
(TNF-α), G-protein coupled receptor (GPCR), Tyrosine
Kinase β, Neurokinin, Rho Kinase, Protein Kinase Mζ
(PKMζ), 3-α-Hydroxysteroid-oxido-reductase (3-α-HSOR),
NADPH oxidase-2 (NOX2), Matrix metalloproteinase-24
(MMP24), Lysophosphatidic acid (LPA), Cytokines, Opioids
and their receptors (κ, δ and µ), Kinins and their receptors
(Kinin B1 and B2 receptor), Protease-activated receptors, Toll
like receptors, Mrg-related GPCR, Prostanoids and
receptors, Interleukin (IL)-1, TNF-alpha, Adrenoceptors,
Glutamate receptors, Gamma-amino butyric acid receptors
(GABA receptors), Ion channels (ligand gated, voltage
gated, Acid-sensing channels, Sodium channels, Calcium
channels), Kinases (PKA, PKC and mitogen-activated
protein kinases (MAPK)), Botulinum toxin and Nitric oxide
etc. are few targets to name [2-16]. This review highlights
the current targets for the treatment of neuropathic pain. In
this review, we have tried to list the important mediators and
have broadly divided them in conventional and non-
conventional targets for treatment of neuropathic pain.
ION CHANNELS
Many ion channels are located at the nociceptor
peripheral terminal, affecting neuronal excitability after
injury and pain sensation [17]. To date, ion channels have
become an important target in search of pain therapies. Ion
channels play a very important role in sensation as they are
responsive to various stimuli. Ion channels consist of
members of multiple gene families. Ion channels are mostly
located at the nociceptor peripheral terminal. On injury, they
excite the neuron which in turn sends the pain signal to the
brain. Voltage gated Na+, Ca2+ channels, transient receptor
potential (TRP) channels, acid sensing ion channels (ASIC),
ligand gated ion channels, P2X, NMDA, AMPA and Kainate
receptors are some of the ion channels reported to be
involved in various pain pathways. The specific contribution
of each ion channels towards nociception and hence pain is
described below.
VOLTAGE GATED ION CHANNELS
Sodium (Na+) Channels
Sodium channels are important for the action potential
generation as they are responsible for rapid depolarization of
the membrane. Till date, ten transcripts encoding for the α-
subunit of Na+ channels have been discovered (Nav1.1-
Nav1.9) [18] which are expressed in different proportions in
sensory neurons. Nav1.1 is mainly expressed in large neurons
while Nav1.6 and Nax are highly expressed in medium and
large neurons whereas Nav1.7, 1.8 and 1.9 are expressed
favorably in selective neurons. Since Na+ channels are
sensitive to tetrodotoxin (TTX); these channels are divided
further into tetrodotoxin sensitive and tetrodotoxin resistant
types. Nav1.3 and 1.7 are TTX-sensitive whereas Nav1.8 and
1.9 are considered as TTX-resistant. Since tetrodotoxin
blocks the ectopic discharges that originate from injured
sensory nerves involved in generation of signals in
neuropathic pain, thus indicates involvement of Nav channels
in the generation of ectopic discharges. This has been
supported by the fact that Nav1.3 and Nax levels are
increased in DRG after nerve injury [19]. On the other hand,
Nav1.8, which is TTX resistant Na+ channel, is
downregulated in injured sensory neurons and upregulated in
un-injured sensitized neurons confirming their involvement
in neuropathic pain pathways [20].
The α subunits of NaV 1.8 and 1.9 are expressed
exclusively in small unmyelinated fibers, whereas the
Nav1.7α subunit is expressed in sensory and sympathetic
neurones. Studies have also indicated that Nav1.7, Nav1.8
and Nav1.9 play important roles in inflammatory pain [21].
Hence it can be concluded that sodium channels are
implicated in neuropathic pain, though it is not clear which
channel isoforms contribute to neuropathic pain.
It has been found that increased density of sodium
channels in the sensory neurons after axonal injury is
associated with abnormal excitability [22]. It has also been
estimated that mRNAs encoding Nav1.1, Nav1.3 and Nav1.6-
1.9 are expressed in sensory neurons. Nav1.1 and 1.6 are also
expressed at high levels in other neurons within CNS while
the distribution of Nav1.3, Nav1.7, Nav1.8 and Nav1.9 is
limited to peripheral nociceptive neurons [23]. Sodium
channels specifically targeted for neuropathic pain therapy
are listed below.
Nav1.3
This channel exhibits fast activation and inactivation
kinetics and rapid recovery from inactivation. Nav1.3
channels have been found to be localized within distal axon
tip in experimental neuromas in rats and humans [24]. This
might explain the ectopic firing within neurons of large
Table 1. Animal Models for Neuropathic Pain. These Animal Models have Helped to Identify Various Mediators and Targets
which are in One or Other Way Involved in the Pathogenesis of Neuropathic Pain
Animal Models for Neuropathic Pain
Nerve Injury Models
Models Mimicking a Particular Disease Condition
Peripheral Nerve Injury
Central Nerve Injury
Neuroma model
Chronic constriction nerve injury
Spinal nerve injury
Partial sciatic nerve injury
Spared nerve injury model
Infra orbital nerve injury
Spinal cord injury
Diabetic neuropathy
Chemotherapy induced Pain

Targets for Neuropathic Pain - A Review CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 65
diameter of Aβ and Aδ fibers of axotomized sensory neurons
[25].
Nav1.7
Nav1.7 is highly expressed in sensory neurons and
ganglia. This channel exhibits fast activation and inactivation
kinetics but comparatively slower than other TTX-sensitive
current. Down regulation of mRNA expression and protein
levels of Nav1.7 has been observed in spinal nerve ligation
model of neuropathic pain [19].
Gain-of-function mutation of SCN9A gene encoding
Nav1.7 has been linked with inherited erythromelalgia (IE).
IE is characterized by hot skin flashes, burning and
paroxysomal extreme pain disorder (PEDP). The mutation
renders Nav1.7 incompletely inactivated, leading to
prolonged persistent currents. Hence the alteration caused by
mutation indicates that Nav1.7 channel is involved in severe
chronic pain pathways.
Nav1.8
Nav1.8 is preferentially expressed in trigeminal neurons
and small diameter sensory neurons. It exhibits slower rate
of depolarized activation and inactivation kinetics. Also it
has been reported that Nav1.8 channels are responsible for
shaping the action potential waveform in nociceptive
neurons [26]. In addition, it has been also reported that
Nav1.8 is not inactivated by cold perception and hence it is
essential for perception of cold pain [27]. Increased Nav1.8
immuno-reactivity in uninjured axotomized sensory neurons
has been reported to be a result of spontaneous firing in
uninjured neurons. Also, primary afferent sensory neurons
containing Nav1.8 contributes to hyperactivity of uninjured
neurons in neuropathic pain [28].
Nav1.9
Nav1.9 is a TTX-resistant channel and is prominently
expressed in small diameter sensory neurons. These channels
exhibit voltage-dependent activation close to resting
membrane potential and steady-state inactivation at
relatively positive potential, hence it can be assumed that
Nav1.9 may prolong the response to sub-threshold
depolarization and generate a persistent current. Nav1.9 gene
is upregulated in inflamed conditions, the expression of
Nav1.9 mRNA and protein are downregulated in axotomized
sensory neurons and in animal models of neuropathic pain
[29]. In addition, not much information is available about the
role of Nav1.9 in neuropathic pain signaling [30]. Hence it is
clear that Nav1.9 is mainly involved in inflammatory pain
and its role in neuropathic pain is still unclear.
Calcium (Ca2+) Channels
Voltage gated Ca2+ channels (VGCCs) are present in
almost every cell which converts biological activity to an
electrical signal. VGCCs are large proteins where α1 subunit
at the center creates the pore, surrounded by auxiliary α2δ, β
and γ subunits [31]. VGCC’s can be divided in two
categories: high-voltage activated (HVA) which include L-,
N-, P/Q- and R-type Ca2+ channels, requiring strong
depolarization for activation, and low voltage activated
(LVA) T-type Ca2+ channels which can be activated by
milder depolarization. L-type Ca2+ channels are mainly
located in CNS whereas P/Q-, N- and R- type Ca2+ channels
mostly are involved in synaptic transmission and T-type Ca2+
channels are responsible for neuronal excitability.
L-Type Calcium Channels
Though, in the CNS, L-type Ca2+ channels are most
efficient in regulating activity-dependent gene expression,
their role in pain has not been well documented [32]. In near
past, L-type calcium channel blockers in combination of
opioids have been shown to have higher antinociceptive
effect than that produced by either of the drugs administered
alone [33]. Another study have reported that knockdown of
Cav1.2 in the spinal dorsal horn reversed the neuropathy-
associated mechanical hypersensitivity and increased
responsiveness of dorsal horn neurons [34].
N-Type Calcium channels
The α1B subunit of the N-type Ca2+ channel is encoded by
the Cav2.2 gene which contains extensive splice variants. It
has been found that the level of α1B is upregulated after
inflammation or nerve injury [35]. The sensitivity of N-type
channels towards toxin blockade increases after nerve injury.
Spinal administration of N-type channel blockers
significantly attenuated hyperalgesia and allodynia in animal
models which clearly indicated that N-type channel play an
important role in neuropathic pain. Furthermore, studies
using α1B knockout mouse have showed that animals
experienced attenuated nociceptive responses following
nerve or tissue injury, thus confirming the role of N-type
Ca2+ channels in neuropathic pain [36]. Some effective N-
type Ca2+ channel blockers include Prialt (synthetic form of
peptide ω-conotoxin MVIIA), AM336 (ω-conotoxin CVID)
and NMED-160.
P/Q-Type Calcium Channels
P/Q-type channels have been reported in epilepsy, ataxia
and migraine. P/Q-type channels are basically distributed at
synaptic terminals at laminae II-VI of the dorsal horn. It has
been established that increased influx in Ca2+ influx through
P/Q-type channels increases the chances of chronic
spreading depression, which might trigger a migraine
episode. These channels have been reported to be involved in
inhibitory neurotransmission in the spinal dorsal horn [37]. It
has been reported that P/Q-type channel blockage at spinal or
supraspinal level produced hypersensitivity [38] but blocking
P/Q-type channel activity at spinal level reduced
hyperalgesia [39]. These studies confirm the involvement of
P/Q-type Ca2+ channels in pain mechanisms.
R-Type Calcium Channels
Although not much information is available on the R-
type calcium channels, it has been reported that the high
voltage-activated Ca2+ channels in peripheral sensory
neurons contribute to pain transmission. Also, it is believed
that R-type Ca2+ channel isoforms are present in nociceptive
DRG neurons and hence R-type channels might be of
potential interest for pain treatment [40].

66 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 Gangadhar et al.
T-Type Calcium Channels
T-type Ca2+ channels are divided as Cav3.1, 3.2 and 3.3.
These channels are primarily located in dendrites and cell
body of the neurons involved in pain. Of the three subtypes,
inhibiting Cav3.2 expression in DRG neurons was found to
attenuate nociceptive response after nerve injury [41].
Similarly it has been found that blocking thalamic T-type
channels results in hypersensitivity to noxious visceral
stimuli suggesting a tonic antinociceptive function of
supraspinal Cav3.1 [42]. These facts suggest that Cav3.2
inhibitor might be a potential drug candidate for the
treatment of neuropathic pain. A major advantage of T-type
channel blocker would be that it will act peripherally; hence
the CNS side effects can be avoided.
Potassium (K+) Channels
Potassium (K+) channels play an important role in
neuronal excitability as they stabilize the membrane potential
by producing hyperpolarizing outward currents. K+ channel
show tremendous structural and functional diversity leading
to a variety of membrane properties in different cells [43].
K+ channels can be divided in four categories: (a) voltage-
gated K+ channels (Kv), (b) inwardly rectifying K+ channels
(Kir), (c) Ca2+- activated K+ channels (also called Maxi-K+
channels) and (d) two-pore K+ channel (also known as
tandem-pore K+ channels or K2P channels). Kv channels open
in response to depolarization and help to shape the action
potential. Kir channels have two trans-membrane domains
and a pore region in between and functions as a part of
signaling pathways. G-protein gated K+ channel is a subtype
of Kir channel, and is activated by Gβγ subunit and mediates
G-protein based cell responses. Ca2+-activated Maxi-K+
channels are gated by intracellular Ca2+levels [44]. K2P
channels can be further categorized as tandem-pore weak
inwardly rectifying K+ channel (TWIK), tandem-pore acid-
sensing K+ channel (TASK), TWIK-related K+ channel
(TREK), and TWIK-related alkaline pH-activated K+
channel (TALK) [45].
Despite K+ channels play an important role in signal
transmission, the link between K+ channel and pain has not
been well understood. Since Kv channel can suppress
nociceptive neurons by hyperpolarizing them, Kv channel is
being highlighted as a target for the treatment of neuropathic
pain [46]. A variety of voltage-gated K+ channels are found
in sensory neurons [47]. Several delayed rectifier type K+
channels have been described in small as well as larger
diameter DRG neurons [48]. Among Kv subtypes, Kv1.4
subunit shows high expression in specific nociceptors [49].
Above studies have also suggested that the increase in
excitability of nociceptor is due to the decrease in Kv1
expression levels in the state of chronic bladder
inflammation.
TRANSIENT RECEPTOR POTENTIAL (TRP)
CHANNELS
Transient receptor potential (TRP) channels are named
after their role in drosophila photo transduction. These
constitute a large family of channels with a wide range of
physiological functions. The structure of TRP channels
relates them to the superfamily of voltage-gated channel
proteins, as they also have six transmembrane domain
segments, a pore region between 5th and 6th transmembrane
domain, and a voltage sensor. In humans, 28 different TRP
genes have been identified and grouped into 6 families:
classical TRP channels (TRPC), vanilloid receptor-related
TRP channels (TRPV), melastatin related or long TRPs
(TRPM), the mucolipins (TRPML), the polycystins (TRPP),
and ankyrin transmembrane protein-related channels (TRPA)
[50]. TRP channels have been associated with specific
sensations like vision, taste, smell, hearing,
mechanosensation thermosensation and pain [51].
Ion channels of TRP involved in thermosensitivity are six
in number. Each of these members of TRP channel family
has different thermal threshold: TRPV4 (>25 ᵒC), TRPV3
(>31 ᵒC), TRPV1 (>43 ᵒC), TRPV2 (>52 ᵒC), TRPM8
(>28 ᵒC) and TRPA1 (>17 ᵒC). TRPV1 (also known as
capsaicin receptor or vanilloid receptor, VR1) has been
found to be involved in thermal nociception. TRPV1 is
activated by heat and capsaicine [52].
In fact, it has been found that capsaicin acts mainly
through TRPV1 [53]. The first TRPV1 was reported to be
mainly expressed in nociceptors and in sensory neurons and
is found to be associated with inflammation and tissue
injury. In native DRG neurons and in heterogeneous system,
the inflammatory mediators, ATP and bradykinin were
reported to potentiate TRPV1 through the P2Y2 and B2
receptors respectively. The other members of TRP family,
TRPV2, TRPM8 and TRPA1 have activation thresholds
within the noxious range of temperature indicating their
possible involvement in thermal nociception [52]. For the
first time, the involvement of TRP channels in the pain
mechanisms was confirmed by cloning vanilloid receptor
TRPV1 [54]. Studies indicated that TRPV1 plays an
important role in the detection and integration of noxious
stimuli [55]. Analysis of TRPV1 gene knockout mice
confirmed that the channel contributes to the detection of
acute painful chemical and thermal stimuli [53, 56]. A
number of subsequent studies have highlighted the role of
other TRP channels in detecting of painful stimuli by
nociceptor neurons.
For neuropathic pain in humans, members of the TRPV
family especially TRPV1 and TRPV4 have been indicated.
Release of inflammatory mediators, low pH, activation of
PKC, protease activated receptor2 (PAR2) and
phosphoinositol 3-kinase respectively might result in thermal
allodynia and hyperalgesia through TRPV1 [57]. Cold
hyperalgesia has been associated with TRPA1, whose
expression in DRG neurons increases in inflammation and
nerve injury. Despite the fact that TRPM8, a cold sensing
channel is found in abundance as TRPA1 knock down by
siRNA strategies suppressed cold hyperalgesia in an animal
model of nerve injury [58]. This indicated the importance of
TRPA1 for the treatment of cold hyperalgesia caused by
inflammation or nerve damage. Recently it has been reported
that activation of TRPM8 in rat models of neuropathic pain
by either cutaneous or intrathecal application of
pharmacological agents or by modest cooling caused
inhibition of sensitized pain response [59].
The analgesic effect of TRPM8 activation relies on group
II/III metabotropic glutamate receptors (mGluRs) as
glutamate released from TRPM8-containing neurons

Targets for Neuropathic Pain - A Review CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 67
suppresses nociceptive inputs. Interestingly, the analgesic
profile of TRPM8 activators seem to be different from the
pronociceptive profile of TRPA1 activators and merits
further investigation on the potential relationship between
TRPM8-mediated analgesia and TRPA1-related cold
hyperalgesia in different subpopulations of sensory neurons.
Understanding of these two antagonistic cold-activated
mechanisms could result in novel strategies for intervention
in chronic sensitized pain states.
LIGAND GATED ION CHANNELS
Purinergic Receptors
Since long, it has been established that adenosine 5’-
triphosphate is released as co-transmitter in nerves of both
central and peripheral nervous system (PNS) [60]. Initially
these receptors were identified as P1 and P2 (corresponding
to adenosine and ATP/ADP, respectively). Four P1 receptor
subtypes A1, A2A, A2B and A3 have been cloned and
characterized. All P1 adenosine receptors couple to G-
protein coupled receptors. Depending on pharmacological
basis, P2 receptors were further divided into P2X and P2Y
receptors. P2X represents family of seven ligand gated ion
channels (P2X1-7) and P2Y belongs to family of eight G-
protein coupled receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11,
P2Y12, P2Y13 and P2Y14). Recent studies have shown that
P2X3 is highly expressed in nociceptive sensory neurons in
DRG [61]. There is enough evidence that many of these
receptors are involved in pain transduction pathways.
Activation of P2X receptors in spinal cord caused allodynia
[62]. Another study reported upgradation of P2X4 receptors
in neuropathic pain conditions [63]. It has been reported that
P2X7 and P2Y12 receptors on microglia are also involved in
neuropathic pain. There are evidence suggesting involvement
of P2X2, P2X3, P2X4 and P2X6 receptors participating in pain
pathway. A review covering the role of purinergic receptors
in pain is available elsewhere [64].
Acid Sensing Ion Channels (ASIC)
A stable pH is required for the normal functioning of
cells. In normal physiological conditions various H+
transport mechanisms maintain the extracellular pH of 7.3
and intracellular pH of 7.0. Pathophysiological conditions
like inflammation, infection, ischemic stroke, neurotrauma,
epileptic seizure or tissue injury, accumulation of lactic acid
due to enhanced anaerobic glucose metabolism and release
of H+ from ATP hydrolysis results in lowering the pH, which
leads to acidosis. Since long, it has been known that
extracellular acidosis would elicit pain [65]. The primary
sensory neurons are the one to detect the local drop in pH.
These types of proton-gated channels can be divided in two
types: transient receptor potential vanilloid receptor 1
(TRPV1) and acid-sensing ion channels (ASICs).
ASICs belong to the voltage-insensitive, amiloride
sensitive epithelial Na+ channel/degenerin family of cation
channels [66] which includes the epithelial Na+ channel
(ENaC), FMRF-amide-activated channel (FaNaC) of
invertebrates, the degenerins (DEG) of Caenorhabditis
elegans, channels in Drosophila melanogaster and orphan
channels (BLINaC and INaC) [67]. ASICs are expressed in
both CNS and PNS. A total of six ASIC’s, encoded by four
genes have been identified. These are ASIC1a, ASIC1b,
ASIC2a, ASIC2b, ASIC3 and ASIC4 [68]. ASIC subunits
are made up of two transmembrane domains (TMI and
TMII) linked by extracellular cysteine-rich loop and
intracellular N and C terminals. Functional ASIC’s are
supposed to be tetrameric assemblies of homomeric or
heteromeric subunits.
Activation of ASICs by change in pH can depolarize the
neurons and generate action potential leading to pain
perception. Decrease in pH has been associated with non-
adapting pain in humans [69]. Likewise cutaneous acid-
induced pain (up to pH 6.0) is likely to be mediated by
ASICs [70]. In addition to ASICs, another ion channel
TRPV1 (capsaicin receptor) contributes to pain mechanisms
by responding from DRG in low range pH conditions (pH
less than 6.0). Almost all ASICs are present in primary
sensory neurons of the vagal, trigeminal and DRG. ASIC1a,
ASIC1b, ASIC2b and ASIC3 are extensively expressed in
small and medium nociceptive neurons and detect noxious
chemical, thermal and high threshold mechanical stimuli
[71]. In CNS, ASIC1a, ASIC2a and ASIC2b are widely
expressed in the brain. Of these, the one which cannot be
activated by protons has been reported in the pituitary gland,
brain, spinal cord and retina [72].
ASIC3 containing channels display a sustained
component, in addition to the transient current that does not
get inactivated in acidic pH. The sustained current involves
different mechanisms depending on the extent of pH change.
It results from the window of overlap between inactivation
and activation of the transient current for small extracellular
acidification (between pH 7.3 and 6.7) [73] but seems to be
independent of the peak current at acidic pH.
ASICs play an essential role in pain sensation. Their
involvement in the development of central sensitization and
pain hypersensitivity is well documented [74]. Spinal dorsal
horn neurons express a high density of harmonic ASIC1a
channels and the expression of these channels was
upregulated by peripheral inflammation [75]. Blocking of
ASIC1a by spinal infusion or by its specific inhibitor PcTx1
or suppression of ASIC1a expression using specific
antisense oligonucleotides markedly attenuated complete
freund’s adjuvant (CFA)-induced thermal and mechanical
hypersenaitivity [76]. In animal models of neuropathic pain,
ASIC3 immunoreactivity in rat DRG neurons was found to
be elevated following lumbar disc herniation [77] while
intrathecal injection of PcTx1 reserved the thermal and
mechanical nociception in rats with chronic constriction
injury (CCI) [78].
Protein Kinase M Zeta
Ca2+ and phospholipid-dependent protein kinases C
(PKC) represent a family of second messenger-dependent
protein kinases. PKC plays a pivotal role in mediating
cellular responses to extracellular stimuli involved in
proliferation, differentiation, apoptosis, and exocytotic
release in a number of non-neuronal systems such as islet
cells, chromaffin cells and paramecium. PKC has also been
implicated in phosphorylation of several neuronal proteins,
which are thought to regulate neurotransmitter release and
establish long-term potentiation in memory formation [79].

68 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 Gangadhar et al.
PKC is not a single enzyme but a family of
serine/threonine kinases. At least 10 isoforms of PKC are
known and are classified by their activation requirements.
PKC activation occurs when plasma membrane receptors
coupled to phospholipase C are activated, releasing
diacylglycerol. The conventional isoforms, α, βI, βII, and γ
are activated by phosphatidylserine, diacylglycerol and Ca2+.
The unconventional isoforms δ, ε, η and θ require
phosphatidylserine and diacylglycerol but do not require
Ca2+. The ζ and λ isoforms are called atypical and require
only phosphatidylserine for activation. Among them, only
protein kinase M zeta (PKMζ) maintains persistent synaptic
changes [80]. PKMζ, has been detected in many regions of
the brain, including the frontal cortex [81]. In the
hippocampus, long term potentiation (LTP) induction
triggers the synthesis of PKMζ and is regulated by other
protein kinases. Recent studies have indicated the role of
PKMζ in (a) the potentiation of postsynaptic receptor
responses, (b) enhancement of the synaptic transmission
exclusively by increasing the number of functional
postsynaptic AMPA receptors, (c) maintenance of late-LTP,
(d) maintenance of potentiation after synaptic tagging, (e)
maintenance of long-term spatial memory storage in the
hippocampus and (f) maintenance of long-term associative
memory storage in the neocortex [82]. Recently, a team of
scientists from University of Toronto reported that PKMζ
maintain pain-induced persistent changes in the mouse
anterior cingulate cortex (ACC). Peripheral nerve injury
showed activation of PKMζ in the ACC and administration
of a selective PKMζ inhibitor, ζ-pseudosubstrate inhibitory
peptide (ZIP), erased synaptic potentiation. Microinjection of
ZIP into the ACC blocked behavioral sensitization,
indicating the role of PKMζ in ACC in the maintainence of
neuropathic pain [83]. Thus, PKMζ inhibitors could be a
promising target for treatment of chronic pain. Till date, two
potent PKMζ inhibitors are extensively used in studies: (a)
ZIP, a selective PKMζ inhibitor (see Fig. 2) and (b)
Chelerythrine which inhibits catalytic domain of PKC (see
Fig. 2).
Rho Kinase
Rho proteins comprise a subfamily of highly conserved
small molecular G-proteins that belong to the Ras
superfamily. In neuronal cells, RhoA is involved in the
guidance and extension of axons and the development and
structural plasticity of dendrites and dendritic spines [84].
Several studies have suggested that RhoA regulates the
stability of dendritic branches in neurons [85]. Like PKMζ,
RhoA has also been shown to play an important role in the
formation of long-term potentiation in hippocampal neurons
[86]. Thus, the activation of RhoA signaling is involved in
the formation of synaptic plasticity in the CNS.
A growing body of evidence indicated that the synaptic
plasticity of dorsal horn neurons contribute to pain
hypersensitivity after strong noxious stimulation [87].
Several intracellular protein kinase cascades mediate the
formation of synaptic plasticity of dorsal horn neurons [88].
Rho/Rho kinase (ROCK) pathway plays an important role in
the development and/or maintenance of chronic pain [89].
There have been reports that intrathecal treatment with the
ROCK inhibitor Y27632 (see Fig. 2) attenuates cold
hyperalgesia [90]. Recently it was also reported that
intrathecal treatment with mevalonate produced thermal
hyperalgesia through the activation of spinal RhoA/ROCK
signaling [91]. In an acute pain model, systemic treatment
with the ROCK inhibitor Y27632 produced an
antinociceptive effect in the hot-plate and acetic acid
writhing tests [92]. Therefore, spinal activation of the
RhoA/ROCK pathway sensitizes nociceptive transmission
and is involved in the development and maintenance of
hyperalgesia. Y27632 is the oldest synthesized and reported
specific inhibitor of Rho-kinase family enzymes. Y27632
inhibits ROCK activity by competitively binding at the ATP
domain. Another novel ROCK inhibitor known is H1152
(see Fig. 2), which is a more specific, stronger and
membrane permeable inhibitor of ROCK with a Ki value of
1.6 nM. Fasudil is also another ROCK inhibitor (see Fig. 2)
which has been widely used in the field of neuropathic pain
research [93]. These inhibitors have a greater specificity for
ROCK, when compared to protein kinase A and PKC.
ANTIDEPRESSANTS
To an extent, pain and depression share common features
(e.g. fatigue and sleep disturbance). But there is no enough
evidence to explain how antidepressants could effectively be
used for pain management. They are believed to exert their
effects through serotonin and norepinephrine receptors. They
also might relieve pain through histamine receptors and by
modulating sodium ion channels [94].
Tricyclic Antidepressants (TCAs)
Since tricyclic antidepressants (amitriptyline, doxepin
and imipramine) have been used in pain management for
decades and being cheaper, they have been considered as the
first choice for the treatment of various types of pain. The
disadvantages include the side effects like arrhythmias,
hypertension, postural hypotension (creates problem to older
adult patients) and last but not the least, there is a potential
risk of lethality with an overdose. Though there is enough
evidence that TCAs are the first choice for the treatment of
neuropathic pain, their side-effects made researchers to
relook for agents that enhance serotonin and norepinephrine
transmission as discussed in the following sections.
Serotonin Norepinephrine Reuptake Inhibitors (SSRIs
and SNRI’s)
Serotonin and noradrenaline, like endogenous opioids
and GABA neurotransmitters modulate the activity in the
nociceptive pathway at the dorsal horn of the spinal cord.
Inhibition of presynaptic reuptake of the monoamines,
serotonin and noradrenaline by antidepressants increases the
levels of these amines in the synaptic clefts and thereby
increasing the pain suppression induced by this system [95].
Selective serotonin reuptake inhibitors (SSRIs) exert
their effect by inhibiting the reuptake of serotonin. SSRIs
along with tricyclic antidepressants are basically used as first
line medications for treatment of neuropathic pain [96].
SSRIs alone have been used as third line of medication to
treat neuropathic pain. Some early clinical trials have shown

Targets for Neuropathic Pain - A Review CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 69
Fig. (2). Structures of important inhibitors acting on various pain pathways. Protein kinase M Zeta inhibitors: (a) z-Pseudo substrate
inhibitory peptide (ZIP) and (b) Chelerythrine; Rho Kinase inhibitors: (c) Y27632, (d) H1152 and (e) Fasudil; Selective serotonin reuptake
inhibitors: (f) Paroxetine, (g) Fluoxetine, (h) Sertraline, (i) Fluvoxamine, (j) Lofepramine, (k) Citalopram and (l) Escitalopram; Serotonin
norepinephrine reuptake inhibitors: (m) Duloxetine and (n) Venlafaxine; Glial inhibitors or glial modifying drugs: (o) Fluorocitrate and (p)
Propentofylline; 5-HT3R antagonist: (q) Ondansetron; Gap junction inhibitor: (r) Carbenoxolone; NGF inhibitor: (s) Acetyl-L-carnitine
(ALCAR) and (t) ALE-0540; protein kinase inhibitor, and Tyrosine kinase inhibitor: (u) 1-NM-PP1.
Myr-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu N+O
O
O
O
N
NHO
H2N
N
S OO
N
NH
S
N
O O
N
NH
N
H
F
O
O
O
H
O
H
N
F F
F
NH
Cl
Cl
O
F
FF
NO
NH2
N
N
O
Cl
N
O
F
N
O
N
F
N
O N
H
S
O
N
OH
O-
O
-O
O
F
OH
O
HO
N
N O
O
N
N
O
N
ON
N
O
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HO
O
H
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HO
HO
O
O
N+
OOH
H
OO
N
OH
H
N+
O-
O
N
N
N
H2N
(a)(b)(c)
(d)(e)(f)
(g)
(h)(i)
(j)(k)
(l)(m)
(n)
(o)
(p)
(q)
(r)
(s)
(t)
(u)

70 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 Gangadhar et al.
beneficial effects for paroxetine and citalopram whereas
fluoxetine failed to show any efficacy [97]. Hence SSRIs
began to replace TCAs for depression, and same is true for
neuropathic pain as compared to TCAs, SSRIs are safer
against overdose and have acceptable adverse effect profile.
SSRIs are non-tricyclic drugs which are characterized by
causing serotonin reuptake inhibitors with no action on
noradrenaline reuptake. Paroxetine, fluoxetine, sertraline,
fluvoxamine maleate, lofepramine, citalopram and
escilatopram are few examples of widely used SSRIs (see
Fig. 2).
Recently a number of trials have been conducted to study
the effects of serotonin norepinephrine reuptake inhibitors
(SNRIs) on painful diabetic neuropathy [98] and painful
polyneuropathy. Duloxetine and venlafaxine (see Fig. 2)
have been studied in peripheral neuropathic pain. Duloxetine
was found to be effective against diabetic peripheral
neuropathy [99] while venlafaxine has shown efficacy in
both diabetic peripheral neuropathy and polyneuropathies. In
a recent review duloxetine have been suggested as an
excellent choice for the treatment of diabetic peripheral
neuropathic pain [100].
Both SSRIs and SNRIs have side effects that include
nausea, vomiting and dyspepsia. But looking at the risk-
benefit ratio, SSRIs and SNRIs appear as better candidates
than TCAs for the treatment of neuropathic pain.
Serotonin Ionotropic Receptor (5HT3R)
Serotonin (5-hydroxy tryptamine, 5-HT) can be divided
into various G-protein coupled metabotrophic receptor
subtypes like 5-HT1A-F, 5-HT2A-C, 5HT4, 5-HT5A, B, 5-
HT6, 5-HT7 and one ionotropic receptor, 5-HT3R [101]. 5-
HT3R is generally involved in the activation of nociceptive
neurons as its expression has been reported in nerve endings
which are involved in nociception. Since long time,
serotonin has been categorized as endogenous pain
producing bioactive molecule which is released from the
activated platelet and enterocromaffin cell and 5-HT3R has
been reported to depolarize 70% C-type neurons and 47% A-
type neurons when administered to Bull Frog DRG [102].
Same is also supported by the fact that 5-HT3R antagonist,
ondansetron (see Fig. 2) reduces neuropathic pain [103].
This clearly indicated that specific 5-HT3R inhibitor design
might result in effective molecules for the treatment of
neuropathic pain.
Astrocytes and Microglia
It has been recently reported that neurons are not the only
cell type involved in chronic pain states. Glial cells,
including astrocytes and microglia are emerging as possible
additional players in the initiation and maintenance of
neuropathic and inflammatory pain [104]. These glial cells
have close interactions with neurons and thus modulate pain
transmission particularly under pathological conditions.
Microglia responds to several neuronal-derived signals after
peripheral nerve injury. These signaling pathways include
ATP and its receptors (P2X and P2Y receptor, discussed in
earlier sections), fractalkine, CX3CR1, monocyte
chemotactic protein (MCP-1) and CCR2. Spinal microglia
responds to ATP via purinergic signaling. Similar to
microglia in the brain, spinal microglia show fast chemotaxis
in response to local application of ATP.
Although glial cells were originally regarded as
supporting cells in the CNS, mounting evidence indicates
that glia actively communicate with neurons and contribute
towards the development of different types of
neurodegenerative diseases. Increasing evidence suggests
that glial cells in the spinal cord play an important role in
pain facilitation. For example, peripheral nerve injury
produces profound changes in glial cells including
morphological changes of microglia and astrocytes and
increased expression of glial markers, such as CD11b, Iba-1
and GFAP. Glial inhibitors or glial modifying drugs such as
fluorocitrate and propentofylline (see Fig. 2) can alter pain
sensitivity. These studies demonstrated the overall role of
glia in regulating pain sensitivity, but it is not clear that
which type of glial cell is involved in pain regulation.
Several lines of evidence suggest that activated astrocytes
are involved in chronic pain symptoms. Hofstetter et al.
(2005) reported about the implantation of neural stem cells
into the injured spinal cord causing allodynic-like
hypersensitivity, which mainly attribute to the conversion of
the stem cells into astrocytes [105]. Astrocytes express
proteases, such as tissue-type plasminogen activator (tPA)
and matrix metalloproteases (MMP) which might be critical
for the cleavage and release of signaling molecules from
astrocytes that are involved in chronic pain [106]. Astrocytes
are characterized by forming gap junction-coupled networks,
which could transmit Ca2+ signaling in the form of
oscillations through the networks [107]. The major structural
components of gap junctions are connexins (Cx) and in the
mammalian nervous system, at least six connexins (i.e.,
Cx26, Cx29, Cx30, Cx32, Cx36, and Cx43) have been
identified. Among them, Cx30 and Cx43 are specifically
expressed by astrocytes [108].
Interestingly, the expression of Cx43 increases markedly
in response to facial nerve lesion, spinal cord injury [109]
and CFA-induced inflammation, indicating a role of Cx in
chronic pain. Inhibition of gap junction function by
carbenoxolone (a non-selective gap junction inhibitor)
produces analgesia in different pain models (see Fig. 2)
[110]. Particularly intrathecal injection of carbenoxolone
reduces sciatic nerve inflammation-induced mechanical
allodynia in the contralateral paw, suggesting a role of
astrocytes network and gap junction in the spread of pain
beyond the injured areas [111].
In addition, astrocytes also express phosphorylated c-Jun
N-terminal kinases (JNK and JNK1) phosphorylated
extracellular signal-regulated kinase (ERK), endothelin
receptor-B, TNFα, basic fibroblast factor (bFGF),
neurokinin-2 receptor, IL-18 receptor and monocyte chemo
attractant protein-1 (MCP-1), in response to nerve injury or
inflammation. Importantly, intrathecal administration of
these molecules has shown to reduce chronic pain symptoms
[112].
IL-1 is a major pro-inflammatory cytokine and is
upregulated in the spinal cord under different chronic pain
conditions [113]. Studies have shown IL-1β upregulation in
astrocytes after nerve injury, hind paw inflammation and
masseter inflammation [114]. IL-1β was also found in

Targets for Neuropathic Pain - A Review CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 71
neurons in the spinal cord [115]. Several lines of evidence
support an important role of IL-1for pain sensitization.
Inhibition of spinal IL-1 signaling with intrathecal IL-1
receptor antagonist or neutralizing antibody has been shown
to alleviate inflammatory and neuropathic pain [115].
Endothelin
Endothelins (ET) are a family (ET-1, ET-2, ET-3) of
ubiquitously expressed peptides involved in the control of
vascular tone. ET-1 causes intense pain via activation of
ETA receptors, modulated by analgesic signals initiated by
ETB receptor activation. ET converting enzymes act on
biologically inactive ET, converting it to active ligand,
which then engages G-protein coupled receptors (ETA and
ETB) to initiate and differentially specify signal transduction
cascades. Knockout models of various components of the ET
axis confirm that ET signaling is critical for normal
development of neural crest and heart [116]. An impressive
literature shows that exogenously administered ET-1 causes
pain and that ETA antagonists decrease ET-1 mediated pain.
ETA antagonism also reduces tactile allodynia in diabetic
rats [117].
ETA receptor antagonism acutely and significantly
reduces thermal and mechanical hyperalgesic responses
within five days after injury. Furthermore, ET-1 and the
ETA receptor are locally upregulated at the site of chronic
constriction injury, suggesting that ET-1 may be involved in
establishing pain after the injury. This suggests that ET-1
could be an important mediator of pain in general and
suggest that ETA antagonist development deserves further
study as a potential novel therapy for neuropathic pain [118].
Kinins and their Receptors
Kninis are important pro-inflammatory mediators
implicated in inflammation and pain following tissue injury.
These bradykinin-related peptides are released in plasma
from damaged tissues in response to tissue injury. Kinins are
formed in injured tissues by proteolytic cleavage of
kininogens under the influence of tissue and plasma
kallikreins. Kinins bind to two types of G-protein coupled
receptors, B1 and B2 . B1 receptors are activated by the
endogenous Kinins lacking the carboxy-terminal Arg residue
whereas B2 receptors are activated by the sequence of
endogenous Kinins BK, and Lys-BK, also known as Kallidin
(KD) [119]. It is generally accepted that the B1 receptor is
expressed in response to inflammation and has low level of
expression in healthy tissue [120]. On the other hand, B2
receptor is widely expressed and normally predominates
over B1 and has higher affinity for bradykinin (BK) and Lys-
BK peptides.
Kinin receptors are expressed in neuronal tissues and are
upregulated in response to painful stimuli. In fact, a recent
study has suggested that Kinin B1 and B2 receptors are
involved in thermal hyperalgesia after partial sciatic nerve
ligation in rats [121]. Another research group has recently
indicated that Kinin B1 receptor activation somehow plays a
major role in neuropathic pain development and proposed
that an oral-selective B1 receptor antagonist will be better in
the treatment and management of chronic pain [122].
Another group of receptors have been reported as peripheral
pro-nociceptive Kinin B1 and B2 receptor-operated
mechanisms contributing significantly to the maintenance of
hind paw cold and mechanical allodynia and thermal
hyperalgesia induced by L5/L6 spinal nerve ligation (SNL)
in rats [123]. Another research group in 2008 has indicated
the role of peripheral and central Kinin B1 receptors in the
brachial plexus avulsion (BPA) model of neuropathic pain.
Like earlier group, they have recommended selective B1
receptor antagonism as valuable tools for management of
neuropathic pain [124]. On the contrary, a recent paper
reported that Kinins are important for nociception associated
with acute short-lasting inflammation but play a minor role
in chronic stages of pain. They reported that knock-out mice
lacking both B1 and B2 receptors didn’t show any change in
response towards baseline nociceptive responses, nocifensive
responses to bradykinin were reduced and acute acetic acid-
induced visceral nociception was reduced by about 70%
[125]. There are reports about the involvement of Kinin B1
and B2 receptors in orofacial thermal hyperalgesia induced
by constriction of infraorbital nerve. The administration of
peptidic B1 and B2 receptor antagonists on the exposed
infraorbital nerve at the time of surgery delayed the
development of thermal hyperalgesia. Hence Kinin B1 and
B2 antagonists might be effective against thermal
hyperalgesia induced by nerve injury. Some recent studies
have also highlighted the role of Kinin B1 receptor in
mediation of nociception and diabetes-induced neuropathic
pain [126]. A recent review has also highlighted the role of
microglial Kinin B1 receptor in diabetic neuropathy and has
indicated that the Kinin B1 receptor might be a good target of
potential value for new medicines in the treatment of chronic
pain [127]. Thus these facts indicate that Kinins play a major
role in neuropathic pain and Kinin B1 antagonists might be
good for the treatment of painful neuropathic pain in future.
Cytokines
Cytokines are low molecular weight glycoproteins that
are secreted mainly but not exclusively by immunological
cells such as T-cells, macrophages and neutrophils. Other
cells that secrete cytokines include keratinocytes, dendritic
cells of the skin, schwann cells and glial cells of the CNS.
They act as intercellular mediators regulating the functions
and differentiation of neighboring cells and are produced in
response to disease, inflammation, or tissue damage.
Cytokine synthesis is prompt and their actions are often
localized with a relatively short half-life. This distinguishes
cytokines from hormones which are constantly produced
with long lasting and more distant effects. The first cytokine
was discovered by Beeson in 1948 as a pyrogenic compound
extracted from polymorphonuclear leucocytes, later known
as IL-1β. Since then, many other cytokines have been
discovered, and these fall into five main categories:
interleukins, interferons, tumor necrosis factors, growth
factors and chemokines. Together, these factors contribute to
the pathogenesis of neuropathic pain. In particular, tumor
necrosis factor alpha (TNF-α), interleukin-1 (IL-1) and
interleukin-6 (IL-6) have been associated with the development
of neuropathic pain in various animal models [3].

72 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 Gangadhar et al.
IGF-1
Insulin-like growth factor 1 (IGF-1), also called
somatomedin C, is a protein that in humans is encoded by
the IGF1 gene. IGF-1 has also been referred to as "sulfation
factor" and its effects were termed "non-suppressible insulin-
like activity" (NSILA) in the 1970s. IGF-1 is a hormone
similar in molecular structure to insulin. It plays an
important role in childhood growth and continues to have
anabolic effects in adults. A synthetic analog of IGF-1,
mecasermin is used for the treatment of growth failure [128].
IGF-1 consists of 70 amino acids in a single chain with three
intramolecular disulfide bridges and has a molecular weight
of 17,066 daltons. Its primary action is mediated by binding
to its specific receptor, the Insulin-like growth factor 1
receptor, abbreviated as IGF1R, present on many cell types.
IGF-1 is one of the most potent natural activators of the
AKT signaling pathway, a stimulator of cell growth and
proliferation and a potent inhibitor of programmed cell
death. IGF-1 is a primary mediator of the effects of growth
hormone (GH), which is released from the anterior pituitary
gland into the blood stream and then stimulates the liver to
produce IGF-1. IGF-1 then stimulates systemic body growth
and has growth-promoting effects on almost every cell in the
body, especially skeletal muscle, cartilage, bone, liver,
kidney, nerves, skin, hematopoietic cell and lungs. In
addition to the insulin-like effects, IGF-1 can also regulate
cell growth and development especially in nerve cells as well
as cellular DNA synthesis.
Decreased IGF-1 activity appears to trigger continual
activation of neurotropic (nerve infecting) viral agents
causing fluctuation and frequent inflammation in the sensory
organs or their nerves. The variability in the sensory system
function does not allow the brain to integrate the different
sensory systems and leads to a dynamic and confusing
clinical state.
Diabetic patients have impaired learning/memory, brain
atrophy and two-fold increased risk of dementia.
Neurotrophic insulin-like growth factor (IGF) levels are
reduced in diabetic patients and rodents and since IGF can
cross the blood-CNS-barrier (B-CNS-B), it was
hypothesized that IGF can prevent cognitive disturbances
independently of hyperglycemia and a generalized catabolic
state [129]. Treatment with IGF1 prevented the behavioral
signs of PDN and reversed the neuronal hyperactivity at the
spinal cord and ventrolateral Periaqueductal gray (PAG) and
the neurochemical changes at the spinal cord and at the
brainstem [130]. Both insulin and IGF1 act as neuronal
growth factors essential for proper neuronal activity.
Therefore, the severity of diabetic neuropathy may be
dependent on the combined loss of insulin and IGF1
activities, beyond the effects of hyperglycemia. These
ligands are also involved in cell survival, synaptogenesis,
neurite (axon and dendrite) outgrowth and nerve
regeneration of neurons. It was reported that systemic IGF1
levels in diabetic patients were slightly lower than those of
non-diabetic individuals, and serum IGF1 levels in diabetic
patients with DN were lower than those in diabetic patients
without DN [131]. A decline in insulin and IGF activity as
well as others neurotrophic molecules like neuronal growth
factor (NGF) and neurotrophin 3 (NT-3) in diabetes may
lead to impaired production and axonal transport of tubulins
and neurofilaments, diminished microtubule and
neurofilament contents and a dwindling of axonal diameters.
Neurotrophins
Neurotrophins (NTs) belong to a family of structurally
and functionally related proteins and they are the subsets of
neurotrophic factors. Neurotrophins are responsible for
diverse actions in the peripheral and CNS. They are
important regulators of neuronal function, affecting neuronal
survival and growth. They are able to regulate cell death and
survival in development as well as in pathophysiologic
states. NTs and their receptors are expressed in areas of the
brain that undergo plasticity, indicating that they are able to
modulate synaptic plasticity. Recently, neurotrophins have
been shown to play significant roles in the development and
transmission of neuropathic pain. The neurotrophins (NTs),
nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4
(NT-4) belong to a family of structurally and functionally
related proteins and are a subset of neurotrophic factors. NTs
are responsible for diverse actions in the developing
peripheral and CNS. Indeed, NTs, once released by target
cells, regulate the type and the number of afferent synapses
by promoting the survival of discrete neuronal sub
populations [132]. They are the important regulators of
neuronal functions, affecting neuronal survival and growth,
regulating differentiation, influencing cell fate choices and
regulating neurite morphology. They are also able to regulate
cell death and survival in the development as well as
pathophysiologic states. NTs and their receptors are
expressed in areas of the brain that undergo plasticity,
indicating that they are able to modulate synaptic plasticity.
Rapid NT-mediated responses like changes in synaptic
activity, probably resulting partly from the activation of
second messengers and/or kinases that in turn affects ion
channel function, neuro-transmitter release, and/or synaptic
structure. Slow NT-mediated responses such as NT-induced
differentiation depend on new gene expression [133]. Mature
NTs are homodimeric proteins derived by proteolytic
cleavage of precursor proteins encoded by different genes.
NTs interact with two categories of cell surface receptors
mediating neurotrophin actions: (a) Trk family (TrkA, TrkB
and TrkC) of high affinity tyrosine kinase receptors and (b)
low-affinity p75 neurotrophin receptor (p75NTR). TrkA
(also known as neurotrophic tyrosine kinase receptor, type 1
[NTRK1]) is the high-affinity receptor for NGF; while for
BDNF and NT-4, it is TrkB; for NT- 3, it is TrkC [134]. All
the members of NT family are able to activate the p75
receptor. Through the activation of these receptors, NTs
activate many intracellular signaling pathways. For example,
NGF binds and dimerizes its receptor TrkA and activates the
receptor's intrinsic tyrosine kinase. Activated TrkA
autophosphorylates several tyrosine residues present in the
receptor's cytoplasmic domain. The phosphotyrosines serve
as binding sites for adapter proteins and kinases such as
phospholipase Cᵧ (PLCᵧ) and phosphatidylinositol-3’ kinase
(PI3K) [135]. These molecules are able to trigger multiple
kinase biochemical cascades that culminate in the
phosphorylation and activation of several transcription
factors i.e. Ras/Raf, the Cdc42/Rac/RhoG protein family,
mitogen and extracellular-regulated kinase (MEK), mitogen-
activated protein kinase (MAPK), extracellular-regulated

Targets for Neuropathic Pain - A Review CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 73
kinase (ERK), which in turn induce direct gene expression
[136]. NGF and BDNF are able to activate cyclic AMP
response element-binding protein (CREB) by a calcium /
calmodulin-dependent kinase IV (CaMKIV)-regulated
pathway [137], suggesting that CREB could play a central
role in mediating NT responses in neurons. Several
neurodegenerative diseases and psychiatric disorders, such as
alzheimer's disease, parkinson's disease, depression and
substance abuse are associated with NT dysregulation [138].
Recently, NTs have been shown to be involved in the
neuronal mechanism underlying neuropathic pain
development and transmission. Due to their important role in
this syndrome, NTs could represent as neuropathic pain
target for the future of pain medicine.
Nerve Growth Factor (NGF)
Various studies have highlighted key roles of NGF in
neuropathic pain conditions [139]. Although originally
discovered as a trophic factor for sympathetic and sensory
neurons, NGF acts as a pathogenic pain mediator [140]. Its
levels are elevated in several painful conditions and its
administration in rats or for that matter in humans result in
pronounced mechanical and thermal hyperalgesia [141]. The
increase in the level of normal firing in the afferent neurons
after nerve injury is due to altered expression of channels like
voltage-gated sodium channels. The mechanisms responsible
for the changes in the channel expression are not yet known, but
NT supply can be involved. Indeed, NGF is able to upregulate
the voltage-gated sodium channel expression in neuropathic
pain states, that it is critically linked to sensitization of
peripheral nociceptors [142]. Recently, in the CCI model of
peripheral neuropathy in rats, it has been demonstrated that
NGF level was increased in the ipsilateral DRG, spinal cord and
PAG. Systematic treatment with the antihyperalgesic and
neuroregenerative compound acetyl-l-carnitine (ALCAR) (see
Fig. 2) was able to bring down NGF levels [143]. In addition,
the NGF expression was increased in the red nucleus of the
brain of neuropathic rats [144], as well as in DRG of rat pups
during postnatal life after CFA-induced peripheral inflammation
[145]. One of the proposed mechanisms of action of NGF might
be due to upregulation of several pain-related genes in the
primary sensory neurons of DRG. Indeed, genes coding for
substance P, calcitonin gene-related peptide, TRPV1, Nav1.8
and Nav1.9 sodium channels and mu-opioid receptor (MOR)
were upregulated by NGF [146]. In addition, the pain-activated
glial cells are important source of NGF [147]. In the neuropathic
pain model obtained by peripheral axotomy, initial NGF levels
declined due to disruption of transport from target tissues. NGF
levels then rebound as satellite glial cells begin to synthesize
NGF and supply it to the neurons. Indeed, NGF and NT-3
synthesis are upregulated in the satellite cells surrounding
neurons in lesioned DRG as early as 48h after nerve injury,
indicating that the satellite cell-derived NTs are involved in the
induction of sympathetic sprouting following peripheral nerve
injury [148]. NGF plays a key role also in post-operative pain.
Surgical trauma induces changes in the CNS pain modulating
mechanisms. Post-operative pain can trigger the central
sensitization of the spinal cord and in turn, can develop into a
chronic neuropathic pain. NGF is released in incised tissue and
contributes to hyperalgesia in incisional pain. NGF mRNA is
increased and the large-molecular weight form of NGF protein
is expressed in the region adjacent to the incision at the plantar
aspect of hind paw in rats [149].
Interestingly, drugs blocking NGF are proving to be
effective in animal models of pain in which the non-steroid
anti-inflammatory drugs (NSAIDs) and opiates have no
effect in pain relief [150]. The development of humanized
monoclonal antibodies to NGF or its TrkA receptor and the
sequestration of NGF using TrkA domain 5 (TrkAd5), a
soluble receptor protein that binds NGF with picomolar
affinity seem to be effective in a number of preclinical
models of pain [151]. Targeting either the extracellular NGF
binding domain of TrkA or probably its intracellular tyrosine
kinase domain with small-molecule TrkA antagonists can be
the next step in the antibody-based therapy. In fact, the anti-
NGF treatment was able to reduce neuropeptide levels and
the nociceptive sensitization in rats was associated with
complex regional pain syndrome type I. According to the
reports, anti-NGF antibodies prevented mechanical
nociceptive sensitization and reduced spinal cord dorsal horn
fos expression and the sciatic nerve neuropeptide content
[152]. Anti-NGF antibodies used in this study were the
TrkA-immunoglobulin G (TrkA-IGG) fusion proteins that
were able to bind NGF, thus blocking the binding of NGF to
the TrkA and p75-NGF receptors and inhibiting TrkA auto-
phosphorylation [153]. Anti-NGF antibodies were able to
reverse the tactile allodynia and thermal hyperalgesia in the
complete Freund's adjuvant-induced hind-paw inflammation,
spinal nerve ligation, chronic constriction injury and
streptozotocin (STZ)-induced neuropathic pain models in
rats and mice. In addition, TrkA receptor also represents a
suitable target for the antibody-based drugs. The anti-TrkA
monoclonal antibody MNAC13 has been shown to possess a
significant anti-allodynic effect on neuropathic pain,
inducing functional recovery in mice subjected to sciatic
nerve ligation [154]. Interestingly, the molecular strategies
directed to blocking TrkA-mediated events were showing
promising results. An intrathecal administration of antisense
oligodeoxy nucleotides to TrkA was able to decrease burn-
induced primary mechanical hyperalgesia in dose-related
manner in rat [155]. A non-peptidic molecule, ALE-0540
(see Fig. 2) inhibits the binding of NGF to TrkA, as well as
signal transduction and biological responses mediated by
TrkA receptors. Administration of ALE-0540 in rats
decreased allodynia in the L5/L6 ligation model of
neuropathic pain [156]. Hence, NGF blockers could be a
new option for the next generation in neuropathic pain drugs.
On the other hand, it has been demonstrated that NGF
could be effective in restoring homeostatic conditions in the
spinal cord and maintaining analgesia in neuropathic pain
animals. Intrathecal NGF administration reduced allodynia,
thermal hyperalgesia, reversed neuro-glial morphological
and the molecular changes occurring in animals suffering
from neuropathic pain. Moreover, one NGF-mimetic peptide
was shown to reduce neuropathic behavior and restore
neuronal function in a rat model of peripheral neuropathic
pain [157]. In a phase-II trial, it has been reported that a
positive effect of recombinant human NGF on neuropathic
pain in HIV-associated sensory neuropathy, though
hyperalgesia at the site of injection was frequent [158].
Taken together these studies it can be said that NGF is one of
the major mediators of neuropathic pain, indicating a new
therapeutic target for pain relief.

74 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 Gangadhar et al.
Brain-Derived Neurotrophic Factor (BDNF)
BDNF shows similar hyperalgesic effects to NGF. BDNF
is involved in the central sensitization and synaptic plasticity
in the spinal cord. It has been shown to contribute to the
development and maintenance of neuropathic pain by
activation of the dorsal horn NR2B-containing NMDA
(NMDA-2B) receptors [159]. Indeed one study suggested
that in the SNL model of neuropathic pain, BDNF
expression is significantly upregulated in the spinal dorsal
horn. The maximal enhancement of BDNF expression
occurred in an early stage (24-48h) after SNL, indicating that
BDNF/TrkB-mediated signaling pathway within the spinal
cord could be involved in the induction of neuropathic pain
in early stage after nerve injury. BDNF expression is also
significantly upregulated in DRG sensory neurons in L5
ventral root transection model of neuropathic pain [160].
This highlights that an increased BDNF expression in DRG
primary sensory neurons and spinal cord dorsal horn could
be contributing to the induction of neuropathic pain. The
primary sensory neurons synthesize BDNF which can be
anterogradely transported to the central terminals of the
primary afferents in the spinal dorsal horn enhanced its local
expression. It has been demonstrated that the oral
administration of protein kinase inhibitor, protein
phosphatase 1 (1-NM-PP1, see Fig. 2), at doses that blocked
phosphorylation of TrkB in the spinal cord, was able to
prevent the development of tissue or nerve injury-induced
heat and mechanical hypersensitivity in mice this clearly
indicated that TrkB signaling is not only an important
contributor to the induction of heat and mechanical
hypersensitivity produced by tissue or nerve injury, but also
is required for the development and persistence of
neuropathic pain [161]. BDNF is involved in the axonal
sprouting of intraspinal serotonergic fibers following the
dorsal root injuries (DRIs). This model of neuropathic pain
results in the permanent disconnection of nerve roots from
the spinal cord and leads to the sensory impairments, loss of
sensation and axonal sprouting of intraspinal serotonergic
fibers. Endogenous BDNF is required for sprouting of
serotonergic axons. Its upregulation is mediated by activated
microglia [162]. Besides serotonergic sprouting, peripheral
nerve injury also resulted in sprouting of the spinal
noradrenergic fibers in rodent dorsal horn. BDNF modulates
the noradrenergic system. Indeed, spinal noradrenergic fibers
were found to be increased in L4-L6 DRG ipsilateral to
injury side and in lumbar spinal cord following nerve injury.
After intrathecal infusion of BDNF antiserum spinal
noradrenergic sprouting was prevented. Researchers suggest
that increased BDNF synthesis and release drive the spinal
noradrenergic sprouting following nerve injury, and this
sprouting may paradoxically increase the capacity for
analgesia in neuropathic pain [163]. One of the proposed
mechanisms of action of BDNF in neuropathic pain can be
through enhanced neuronal sensitivity to painful stimuli and
an increased co-expression of thermo-TRP channels. Indeed,
it has been demonstrated that BDNF is able to regulate the
pattern of expression and the level of activity of the
transducer channel TRPV1 [164], which is a well-known
receptor that has been implicated in the mechanical,
chemical and thermal nociceptive stimuli transmission. It is
noteworthy that TRPV1 is also regulated by NGF [165].
Another possible mechanism is the involvement of microglia
in pain development. In fact, in response to the peripheral
nerve injury, the spinal cord activated microglia, P2X(4)
receptors (P2X(4)R) are overexpressed. Activated P2X(4)R
leads to the release of BDNF from microglia [166].
An antibody-based therapy for NGF showed good results
for targeting BDNF. Indeed, anti-BDNF antibodies were
able to postpone the mechanical hyper-nociception in mice
with brachial plexus avulsion pain model [167]. Repeated
intrathecal pre-treatment with specific anti-BDNF antibodies
was able to abolish thermal hyperalgesia induced by nerve
ligation in mice. Additionally, the thermal hyperalgesia was
completely suppressed by a repeated intrathecal injection of
specific antibody to its full-length TrkB. On the other hand,
recombinant adeno-associated viral vector-mediated
overexpression of BDNF reversed the pain-like behavior in
neuropathic rats, suggesting that by changing the levels of
neurotrophins in the spinal cord micro-environment
following nerve injury, it is possible to recover normal
function [168].
Neurotrophin-3
Neurotrophin-3 (NT-3) has been found to have
antagonistic effects to NGF in the pain processing, through
negative modulation of NGF receptor expression and
associated nociceptive phenotype in intact neurons. It has
been demonstrated that NT-3 reduces overexpression of
Nav1.8 and Nav1.9 channels in DRG neurons of the
neuropathic rats. As mentioned above, NT-3 was able to
interact with the TrkC receptor, but it also possessed the
ability to signal via a TrkA receptor. Interestingly, NT-3 was
able to cause tyrosine-phosphorylation of the glial cell line-
derived neurotrophic factor (GDNF)/Ret receptor and
activation of PI3-kinase/Akt pathway [169]. In addition, NT-
3 was able to reverse the CCI-induced thermal hyperalgesia
through downregulation of TRPV1 receptor expression
[170]. Moreover, NT-3 is able to down regulate the
potassium Kv channel of gene expression in DRG neurons
following the nerve injury [171]. The role played by NT-3 in
neuropathic pain is still not very clear. It has been proposed
that NT-3 can be involved in a long-term change of neuronal
excitability. Indeed, it has been proved that NT-3 promotes
extensive growth of lesioned axons in rat crushed dorsal
columns [172]. Neurotrophic factor delivered by an
adenovirus-based gene therapy has been proposed to be a
promising strategy for the prevention of neuropathic pain
[173]. Recombinant adenovirus encoding NT-3 when
injected intramuscularly in rats with STZ -induced diabetes
showed decreased denervation [174]. Taken together these
findings are consistent with the analgesic role of NT-3.
Conversely, intrathecal administration of NT-3 antisense
oligonucleotides attenuated nerve injury-induced sprouting
and allodynia [175].
Neurotrophin-4
The role of neurotrophin-4 (NT-4) in neuropathic pain is
not very clear. NT-4 is synthesized by DRG and expressed in
the rat spinal cord. It is a ligand of the TrkB tyrosine kinase
receptor, but mediates diverse effects in relation to BDNF
[176]. It has also been demonstrated that repeated injections
of a specific antibody to NT-4 failed to reverse the thermal

Targets for Neuropathic Pain - A Review CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 75
hyperalgesia caused by sciatic nerve ligation in mice. These
results indicate that NT-4 can be an essential component of
nociceptive processing. Indeed, administration of NT-4 to
injured nerves improved their regeneration potential and
affected the axon guidance [177]. In addition, NT-4 drives
peripheral nerve regeneration through regulation of the
expression of myelin-associated glycoprotein, myelin basic
protein, and low-molecular-weight neurofilament protein
[178]. However, further studies are needed to better elucidate
the exact role of NT-4 in a neuropathic pain.
Tissue Necrosis Factor-Alpha (TNF-α)
TNF-α belongs to a superfamily of ligand/receptor
proteins called the tumor necrosis factor/tumor necrosis
factor receptor superfamily proteins (TNF/TNFR SFP).
TNF-α possess a trimeric symmetry with a structural motif
called the TNF homology domain (THD), which is shared
with all other members of the TNF proteins. This THD binds
to the cysteine-rich domains (CRDs) of the TNF receptors
(TNFRs) and variations of these CRDs lead to heterogeneity
of the TNFRs. Several lines of evidence suggest that
proinflammatory cytokines such as TNF are released into the
local milieu after injury and these may initiate the underlying
cascade facilitated processing. TNFRs are either
constitutively expressed (TNFR1, p55-R) or inducible
(TNFR2, p75-R). TNF-α has been detected at CCI injury site
in rats and shows temporal upregulation. TNF-α is located
mainly in macrophages and Schwann cells and is detected by
immuno-reactive staining. Similarly, there is local
upregulation of both TNFR1 and TNFR2 as injured neurons
undergo Wallerian degeneration, albeit at differential rates.
TNF-α enhances the TTX-resistant Na+ current in cultured
DRG cells from wild-type but not from TNFR1-knockout
mice and such current is abolished by a p38-MAPK
inhibitor; implying that TNF-α acts via TNFR1 and activates
TTX-R Na+ channels via the p38 MAPK.
Studies have shown that after nerve injury, TNF
expression increases in dorsal root ganglia (DRG) [179] and
spinal cord [180]. The inhibition of TNF reduced the
hyperalgesia associated with two models of neuropathic
pain: CCI and partial nerve transection [181].
LYSOPHOSPHATIDIC ACID (LPA)
As the name suggests, LPA is identical in structure to
phosphatidic acid (PA), except that LPA has a single acyl
chain. The functions of LPA are extensive and include roles
in development, but individual cellular responses vary
widely. Specific effects are determined by the local
concentration of LPA and on the receptors expressed by a
cell.
Lysophospholipids have recently emerged as important
influences on normal nervous system development. It is a
simple phospholipid that can act as an extracellular signal
through G-protein-coupled receptors [182]. During
development, LPA1 is expressed in neural progenitor cells
suggesting a regulatory function in neurogenesis. In vivo
LPA1 expression has been detected in the hippocampus
wherein it seems to be predominantly restricted to
oligodendrocytes while only hardly detectable levels of
LPA1 mRNA were found in neurons. However, a reasonable
level of neuronal hippocampal LPA1 expression has been
demonstrated under ex vivo circumstances or in immortalized
hippocampal progenitor cells. Exogenous delivery of LPA
has demonstrated LPA1 receptor mediated functions
including morphophysiological changes in neural
progenitors. Likewise, LPA delivery to hippocampal neurons
is known to increase the tyrosine phosphorylation of Focal
adhesion kinase (FAK), regulate cell death, mimic
neurotrophic effects or mediate synaptic changes associated
with spatial memory [183]. Effects of LPA1 loss on inter-
neuron-mediated rhythms in vivo and overexpression gain on
synapse formation have been reported in the hippocampus
[184]. In addition, the presence of modulators of LPA
activity has been demonstrated in the adult hippocampus.
LPA released from injury tissue and transient receptor
potential vanilloid 1 (TRPV1) receptor are implicated in the
induction of chronic pain.
LPA may also be a crucial factor in the initiation of
neuropathic pain mediated by demyelination of peripheral
nerves via activation of LPA receptor. Six subtypes of LPA
receptor are reported (LPA1-6), and all are G-protein-
coupled receptors. Three endothelial differentiation gene
(EDG) family of G-protein-coupled receptors, EDG-2, EDG-
4 and EDG-7 were identified as LPA receptors successively
and were named LPA1-3 respectively. Similarly p2y9 or
GPR23, GPR92 and GPR87 were also identified as LPA
receptors, named as LPA4-6 respectively. Among the six
subtypes, LPA1 receptor is the main subtype expressed in
DRG. LPA1 is capable of interacting with three major G-
protein families, Gi, Gq, and G12; resulting in the activation
of their downstream cascades: mitogen-activated protein
kinase (MAPK), protein kinase C (PKC) and Rho (a small
GTP-binding protein)-Rho kinase, while inhibiting protein
kinase A (PKA) pathway. Several studies have demonstrated
that LPA1 participates in the development of neuropathic
pain through the Rho pathway.
Phosphorylated cAMP Response Element Binding
Proteins (pCREB) Expression Through LPA5 is
Associated with Neuropathic Pain
In the spinal cord dorsal horn, phosphorylated cAMP
response element binding proteins (pCREB) were found to
be upregulated in two different models of neuropathic pain:
CCI and and partial sciatic nerve ligation (PSNL) [185].
Furthermore, downregulation of pCREB was found to be
associated with anti-hyperalgesic effects [186] but the
exposure of LPA5 mutants to LPA stimulation resulted in
increased cellular cAMP levels. This result could help to
predict whether the loss of LPA5 can affect pCREB
expression in the spinal cord dorsal horn after nerve injury.
Ipsilateral-to-contralateral ratio of pCREB expression in the
spinal cord dorsal horn Laminae I-II was markedly reduced
in LPA5 null mice as compared to wildtype control six days
after PSNL. Furthermore, double-labeling of neurons for
LPA5 mRNA and pCREB protein demonstrated co-
localization of both molecules in the same cell, which
suggests that LPA5 directly affects pCREB activation.
Among LPA5 positive cells, 95.4±3.6% were also pCREB
positive, while 83.4±6.0% of pCREB positive cells were
LPA5 positive. This strongly suggested that in the absence of
LPA5, upregulation of pCREB in response to PSNL is

76 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 Gangadhar et al.
abrogated, hence offering protection from neuropathic pain.
Additionally, this effect was not seen in LPA1 null mice,
demonstrating that a different mechanism was involved
[187].
Involvement of LPA1-Signaling in Nerve Injury-Induced
Neuropathic Pain
LPA acts through G-protein-coupled LPA receptors;
LPA1, LPA2, LPA3 and LPA4. Each exhibits different G-
protein interactions. However, only the LPA1 receptor gene
is expressed in DRG neurons. LPA1-null mice reversed LPA-
induced demyelination, as well as mechanical allodynia.
Furthermore, LPA1 receptor-mediated demyelination was
evidenced in the DRG after LPA injection or peripheral
nerve injury by downregulation of myelin-associated
proteins, such as myelin basic protein and peripheral myelin
protein 22 kDa (PMP22). These changes were identical to
nerve injury changes associated with neuropathic pain.
Indeed, LPA1-null mice reversed nerve injury-induced
neuropathic pain and demyelination, as well downregulation
of myelin proteins and upregulation of A-fiber Cav α2δ-1
and spinal PKCγ [188]. Activation of the G-protein-coupled
LPA1 and Rho-ROCK pathway is required for the initiation
and development of neuropathic pain [89].
3α-Hydroxysteroidoxido-Reductase (3α-HSOR)
3α-HSOR is a member of aldo-ketoreductase superfamily
which catalyzes the synthesis and bioavailability of 3a, 5a-
neurosteroids as allopregnanolone (3α,5α-THP). It activates
GABAA receptors and blocks T-type calcium channels
involved in pain mechanisms. Biological functions of 3α-
HSOR include its enzymatic activity in 3 metabolic
pathways, bile acid biosynthesis, c21-steroid hormone
metabolism and metabolism of androgen and oestrogen. 3α
HSOR catalyzes the chemical reaction which involves
conversion of androsterone to 5α-androstane-3, 17-dione and
NAD(P)H, proton in presence of NAD(P)+ [189].
It has been believed that 3α-HSOR is a cellular target and its
modulation in DRG may contribute to suppress pain due to
peripheral nerve injury. Characterization of neurosteroids
demonstrated 3α,5α-THP production in DRG. Reverse
transcription and real-time polymerase chain reaction revealed
that 3α-HSOR mRNA concentration in CCI-rat ipsilateral DRG
was 5-fold higher than in contralateral DRG, and also 4- to 6-
times more than sham-operated or naive rat DRG. Western
blotting confirmed increased 3α-HSOR protein levels in CCI-rat
ipsilateral DRG and double immune-labeling showed that 3α-
HSOR overexpression in DRG neurons but not in glia.
Functional plasticity of 3α-HSOR leading to increased 3α,5α-
THP production was evidenced in CCI-rat DRG. Interestingly,
behavioral and molecular time-course investigations revealed
that 3α-HSOR gene upregulation was correlated to pain
development. Most importantly, in vivo knockdown of 3α-
HSOR expression in healthy rat DRG using 6-
carboxyfluorescein-3α-HSOR-siRNA exacerbated thermal and
mechanical pain perceptions [190].
Neuropathy Target Esterase
Neuropathy Target Esterase (NTE), is a membrane-
bound protein found in neurons of vertebrates and is also
known as patatin-like phospholipase domain-containing
protein 6 (PNPLA6) [191]. It is a phospholipase that
deacetylates intracellular phosphatidylcholine to produce
glycerol-phosphocholine. It is believed to function in neurite
outgrowth and process elongation during neuronal
differentiation. The protein is anchored to cytoplasmic face
of the endoplasmic reticulum in both neurons and non-
neuronal cells [192]. It has been shown to be necessary for
embryonic development in mice and is believed to be
involved in cell-signaling pathways and lipid trafficking.
NTE has serine esterase activity and can hydrolyze ester,
peptide, and amide bonds. The nucleophilic serine residue
(active site) of NTE attacks the carbonyl carbon atom of the
substrate forming a covalent acyl-enzyme intermediate
which is subsequently hydrolyzed. The esterase activity of
NTE is susceptible to covalent inhibition by
organophosphorus esters (OPs) with which it forms an
analogous phosphyl-enzyme intermediate. Irreversible
binding of some compounds to the active serine site results
in a debilitating neural disease known as OP-induced
delayed neuropathy (OPIDN). Signs of OPIDN include
flaccid paralysis of the lower limbs, which becomes evident
two to three weeks after exposure to neuropathic OPs.
Recovery from this disease is usually poor, and there is no
specific treatment. In addition, mutations in the NTE gene
have been linked to motor neuron disease [193].
Matrix Metalloproteinases (MMPs)
Matrix metalloproteinases (MMPs) are identified as zinc
dependent endopeptidases that play essential role in a wide
range of proteolytic processes. So far over twenty members
have been identified in this family. These have been
implicated in the generation of neuroinflammation via
cleavage of extracellular matrix proteins and activation of
proinflammatory cytokines and chemokines, which is their
most prominent function. MMPs are also involved in cellular
differentiation, migration, signaling, survival, and apoptosis
by acting on additional substrates such as proteinases,
chemotactic factors, growth factors, cell surface receptors
and cell adhesion molecules [194].
Most of these MMPs are secreted molecules but some are
transmembrane and glycosyl-phosphatidylinositol-anchored
membrane proteins. MMPs are categorized into four groups
based on their structure and substrate specificity: (a)
Collagenases (MMP-1, MMP-8, MMP-13, and MMP-18),
(b) Gelatinases (MMP-2 and MMP-9), (c) Stromelysins
(MMP-3, MMP-10, and -11), (d) Membrane-type MMPs
(MMP-14 or MT1-MMP, MMP-15 or MT2-MMP, MMP-16
or MT3-MMP, MMP-17 or MT4-MMP, MMP-24 or MT5-
MMP, and MMP-25 or MT6-MMP), and other MMPs.
These proteases normally are not expressed constitutively
but their upregulation was identified when cytokines,
chemokines, growth factors, ECM components, and other
transcriptional regulators act upon the cell [195]. Mostly these
proteases are secreted or translated as inactive precursors that
subsequently require conversion to active enzymes under the
influence of factors such as other MMPs, serine proteases or
free radicals.
The activity of released MMPs is controlled endogenously
by many factors including four physiological tissue inhibitors of
metalloproteinases (TIMPs; TIMP-1, -2, -3, and -4). Recent

Targets for Neuropathic Pain - A Review CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 1 77
studies have shown that these proteases have a critical role in
inflammation through their endopeptidase activity on
extracellular matrix proteins, cytokines, and chemokines [196].
MMPs are also identified as essential factors which are involved
in generation of neuroinflammation, which is believed to
underlie the progression of the disease neuropathy.
There are many proposed neuronal pathways through which
neuropathic pain is believed to progress but these MMPs are
thought to be associated with non-neuronal pain pathways.
Extensive studies are made on temporal and cellular profiles of
MMP expression in the injured spinal cord and the most
important MMPs which are thought to propagate and maintain
neuropathic pain are identified as MMP-2, MMP-9 and MMP-
24 [197].
Increased levels of MMP-2 and MMP-9 immunoreactivity
was found in nerve tissue in chronic inflammatory
demyelinating polyneuropathy (CIDP) and non-systemic
vasculitic neuropathy (NSVN), compared to non-inflammatory
neuropathies (NINs) [198]. Biopsy tissue analyses have shown
the importance of MMP in the pathogenesis of neuropathy.
Recent microarray studies have emphasized the importance
of MMPs for neuropathic pain. Among the four types
gelatinases have been extensively studied due to availability of
gelatinase zymography. Since these proteases are involved in
inflammation, MMP inhibitors have been developed (SB-3CT:
a selective inhibitor of MMP-2 and MMP-9, and reversible
MMP-14 inhibitor, minocycline: a neuroprotective MMP-9
inhibitor) to target different kinds of inflammation-related
diseases such as arthritis, atherosclerosis, periodontitis, and
cancer [199]. Thus all the studies employing recent techniques
such as zymography, gene expression, microarray etc. confirm
the involvement of MMPs in neuroinflammation, propagation
and maintenance of neuropathic pain so further research on non-
neuronal pathways involving MMPs is required for better
understanding of neuropathic pain.
Prospects for New Pain Therapeutics
In last few decades, much effort has been directed
towards understanding the mechanisms of neuropathic pain.
As illustrated above, there is now abundant evidence for
various promising targets which have already shown a
significant degree of efficacy in terms of chronic pain. But
none of these targets have shown complete pain relief in long
term, which in turn underscores the need of newer and better
pain therapeutics. Hence further studies are required to make
sure that these new therapeutic targets possess better efficacy
and lesser side effects.
LIST OF ABBREVIATIONS
3α-HSOR = 3 alpha-hydroxysteroid oxidoreductase
AKT = AKT protein kinase, also known as protein
kinase B
ATP = Adenosine triphosphate
BDNF = Brain derived neurotrophic factor
BK = Bradykinin
BPA = Brachial plexus avulsion
CAMP = Cyclic adenosine monophosphate
CCI = Chronic constriction injury
CFA = Complete Freund’s adjuvant
CNS = Central nervous system
DEG = Degenerin
DRG = Dorsal root ganglia
ERK = Extracellular signal regulated kinase
GABA = Gamma aminobutyric acid
GDNF = Glial cell line derived neurotrophic factor
IASP = International association for the study of pain
IGF = Insulin like growth factor
LPA = Lysophosphatidic acid
MAPK = Mitogen activated protein kinase
MMP = Matrix metalloproteinase
NGF = Nerve growth factor
NMDA = N-methyl D-aspartate
NTE = Neuropathy target esterase
PCREB = Phosphorylated cAMP (Adenosine 3'5' Cyclic
Monophosphate)-Response element binding
protein
PKC = Protein kinase C
PKMζ = Protein kinase M zeta
PSNL = Partial sciatic nerve ligation
RhoA = Ras homolog gene family, member A
ROCK = Rho-associated protein kinase
SNRI = Serotonin norepinephrine reuptake inhibitor
SSRI = Selective serotonin reuptake inhibitor
TCA’s = Tricyclic antidepressants
THP = Tetrahydroprogesterone
TNF-α = Tumor necrosis factor alpha
TRP = Transient receptor potential
TRPA = Transient receptor potential-ankyrin-trans-
membrane
TRPM = Transient receptor potential melastatin
TRPV = Transient receptor potential vanilloid
TTX = Tetrodotoxin
TWIK = Tandem pore weak inwardly rectifying K+
channel
VGCC = Voltage gated calcium channels
ZIP = z-Pseudo substrate inhibitory peptide
COMPETING FINANCIAL INTEREST
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
Declared none.
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Received: January 6, 2013 Revised: March 20, 2013 Accepted: March 20, 2013
PMID: 24152326