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Archives • 2015 • vol.3 • 33-43
AMAZONIAN PLANT EXTRACT BIRM REVERSES CHRONIC NEUROPATHIC
PAIN IN RAT SCIATIC NERVE CHRONIC CONSTRICTION INJURY MODEL
Ravalji, M.1, Buch, P.1, Uggini, G. K.1, Awasthi, A.2, Cevallos-Arellano, E.3; Balakrishnan, S.1*
1Department of Zoology, The M. S. University of Baroda, Vadodara 390 002, Gujarat, India
2Division of Biology, GVK Biosciences Pvt. Ltd., Hyderabad 500 076, Telangana, India
3BIRM Inc, Quito, Ecuador
*suved9@hotmail.com
Abstract
Neuropathic pain condition remains poorly managed by currently available therapeutics. There is therefore a
dire need for development of efficacious therapeutics with minimal side effects. BIRM (Biological Immune
Response Modulator), an extract of Amazonian plant Solanum dulcamara, consumed as a dietary
supplement by natives in Ecuador, is considered as a natural remedy for a number of ailments (AIDS and
Cancer, among others). The aim of the current study was to test the efficacy of BIRM in in vivo neuropathic
pain model to elucidate its anti-neuroinflammatory potential. Rats subjected to chronic constriction injury
(CCI) were divided into CCI-control, CCI-Gabapentin and CCI-BIRM groups along with a normal control group.
BIRM was administered orally (4 ml/kg, daily) to animals of CCI-BIRM group from day 14 post surgery till day
28. Repeated oral administration of BIRM inhibited CCI-induced mechanical allodynia and thermal
hyperalgesia. It also inhibited CCI-induced activation of microglial cells and upregulation of COX-2 and TNF-α
in the dorsal horn of the lumbar spinal cord. These data indicate that the marketed formulation BIRM, has
anti-neuroinflammatory and anti-nociceptive properties in neuropathic rats and can serve as an adjuvant to
standard therapy or as a stand-alone therapeutic agent for the treatment of neuropathic pain disorders.
Keywords: Biological Immune Response Modulator (BIRM), neuropathy, neuroinflammation, microglia cells.
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December 30, 2015
Introduction
Neuropathic pain is a chronic pain condition and is
heterogeneous in nature. It is considered to arise
from damage to nerves due to tumors, diabetic
neuropathy, herpes zoster, complex regional pain
syndrome, AIDS, sclerosis multiplex, hypoxia or
stroke [1] and occurs worldwide. It greatly impairs
quality of life, and has a high economic impact on
society. The Institute of Medicine reports that at
least 116 million American adults suffer from
chronic pain and estimates for people suffering
from neuropathic pain are as high as 17.9% [2].
Symptoms of neuropathic pain are often severely
debilitating such as allodynia, hyperalgesia,
spontaneous pain, as well as behavioral disabilities.
Several animal models mimicking peripheral nerve
injury have been developed to study neuropathic
pain. Most widely used animal models are chronic
constriction injury (CCI) of sciatic nerve [3], partial
ligation of the sciatic nerve (PNL) [4] and ligation of
one or more of the spinal nerves (SNL). Studies
carried out using these animal models in last
decade provides evidence of interactions between
neurons, inflammatory immune and immune like
glia cells, inflammatory cytokines and chemokines.
Peripheral nerve injury provokes a reaction from
the immune system and has been observed at
various anatomical locations including the injured
nerve, the dorsal root ganglia (DRG), the spinal cord
and supraspinal sites associated with pain pathways
[5]. Emerging lines of evidence have revealed that
changes also occur in spinal microglia, the immune
cells of the central nervous system [6]. Activation of
microglia is a major feature of neuropathic pain and
growing evidence suggests that microglia have a
causal role in pathogenesis of persistent
neuropathic pain and hence a detailed study of the
microglial cells and its exact role in central pain
seems necessary.
Till date, Neuropathic pain remains a poorly
managed pathological condition by currently
available therapeutics. Current treatments available
for neuropathic pain indicate general insensitivity to
non-steroidal anti-inflammatory drugs and relative
resistance to opioids. These treatments have
untoward side effects when given at higher doses to
obtain adequate analgesia [7]. Researchers around
the globe are looking for alternate treatment which
can offer adequate analgesia devoid of severe side
effects. Under these circumstances, BIRM
(Biological Immune Response Modulator) seems to
be promising herbal formulation. BIRM is an oral
solution which has been formulated from extracts
of Amazonian plant Solanum dulcamara by a
physician E. Cevallos-Arellano, native of Ecuador. This
formulation is considered as a natural remedy for
number of ailments (AIDS, cancer) and is consumed
as a dietary supplement by Ecuadorian native
population [8].
Earlier studies using BIRM have shown anti-
metastasis properties in in vivo prostate cancer
model [9] and inhibition of PGE2production by COX1
and COX2 [10]. Recently, studies performed using
BIRM in preclinical animal models of pain and
inflammation exhibit its analgesic and anti-
inflammatory properties [11]. Based on these data
available, we found it good enough to explore
undiscovered benefits of BIRM and study it
systematically for assessing its anti-nociceptive effect
in neuropathic pain conditions.
Methods
Animals and housing conditions
Male Wistar rats (180-210 g) were procured from
CPCSEA and AAALAC approved Vivarium Facility at
GVK Biosciences Pvt. Ltd., Hyderabad, India. They
were allowed to acclimatize for a minimum duration
of one week prior to surgical intervention. They were
housed in groups of three in polypropylene cages
under ambient conditions prior to surgery and
housed individually post-surgery. Room temperature
and humidity were maintained at 22–25°C and 65-
70%, respectively. 12 h light/dark cycle was
maintained. Standard laboratory rodent diet and
portable drinking water were provided ad libitum.To
prevent wound infection after a surgical procedure,
the surgical area was dusted with streptomycin
before suturing the incision in all the animals.
Experimental protocols were approved by IAEC
(Institutional Animal Ethics Committee) of GVK
Biosciences Pvt. Ltd. according to CPCSEA
(Committee for the purpose of Control and
Supervision of Experiments of Animals), India
[1125/PO/c/CPCSEA/019(2012)] All animal
procedures were performed in accordance with
guidelines of CPCSEA.
Chronic constriction injury
The method described by Bennett and Xie [2] was
generally followed. Rats were anesthetized with
gaseous anesthesia Isoflurane (Baxter, Germany).
The right common sciatic nerve was exposed at the
level of the mid-thigh by blunt dissection through the
biceps femoris.
Proximal to the sciatic’s trifurcation, about 12 mm of
nerve was freed of adhering tissue and four ligatures
(chromic catgut, Johnson & Johnson) were tied
loosely around it with an interval of about 1 mm
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PhOL Mital Ravalji et al 34 (33-43)
among ligatures. The length of nerve thus affected
was 6-8 mm long. Great care was taken to tie the
ligatures such that the diameter of the nerve was
just barely constricted. The desired degree of
constriction retarded, but did not arrest, circulation
through the superficial epineural vasculature. The
incision was closed in layers.
Test compound and treatment regimen
BIRM was a gift from BIRM Inc. (Quito, Ecuador). It
is an aqueous extract of dried roots of a plant S.
dulcamara (family Solanaceae) grown in Ecuador,
and marketed as a greenish-brown suspension with
a mild bittersweet smell. In the present study, BIRM
samples from same lot number were used and it
was clarified by centrifugation at 10,000 g prior to
usage as described by Dandekar et al. [9].
Gabapentin (Sigma Aldrich, USA) was used as a
standard reference. Though the recommended
minimum dose of BIRM for human consumption, as
per the container label, is 4 ml/day, we used BIRM
at 4 ml/kg dose based on dose range finding study
in in vivo experiments (acute inflammatory models).
The commercially available formulation was directly
administered without further dilution at 4 ml/kg
b.wt. BIRM when administered orally at 4 ml/kg
dose level was found to be well tolerated and found
no observable systemic toxicity in rodents.
Experiment was performed in two sets (n=4 per
group in each set). Data obtained from both sets
was collated and is represented hereby as one
experiment. Surgically operated CCI rats were
randomly selected after assessment of mechanical
allodynia and divided into four groups (n=8 per
group). Drug testing was initiated on day 14 post
surgery. Animals from Normal Control (NC) and CCI
operated Control (VC-CCI) were orally administered
distilled water throughout the study duration.
Animals from BIRM –CCI operated group (BIRM-CCI)
were administered BIRM daily at 4 ml/kg dose
volume (day 14-day 28 post surgery) through oral
route. Gabapentin (30 mg/kg, p.o.) was
administered once on day 14,21 and 28 post
surgery.
Neuropathic pain measurements
Mechanical Allodynia
Mechanical allodynia was assessed by modified
Dixon’s Up and Down method using a set of von-
frey filaments (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0 and
15.0 g).It was assessed at 3hr post first dose on
day 14,21 and day 28 post surgery. The pattern of
positive and negative responses was tabulated
using the convention, X = withdrawal; O = no
withdrawal and the 50% response threshold was
interpolated using the method followed by Chaplan
et al [12].
Thermal Hyperalgesia
Thermal response was determined by measuring
hind paw withdrawal latency of affected paw
employing Hargreaves’ plantar test.
Sample Preparation
On day 28 post surgery (14 days post treatment), the
rats from NC, CCI-VC and CCI-BIRM were deeply
anesthetized with isoflurane 5% and immediately
perfused intracardially with 400-500 ml of cold
phosphate buffered saline (0.01 M, pH -7.4) followed
by 2% paraformaldehyde in 0.01 M phosphate buffer
(pH-7.4) through the ascending aorta. Then their
lumbar spinal cords (L4-L6 region) were quickly
removed. From another set of four animals from
each group, the lumbar spinal cord tissues for RT-PCR
and western blot analysis were collected and
immediately stored at -80°C until analysis.
Western blot –expression of Iba-1 protein –
microglia cell marker
Protein was resolved on 15% polyacrylamide gel
followed by transfer on to nitrocellulose membrane.
Immunoprobing of Iba-1 protein was by Anti-Iba-1
(AbCam, ab5076) used at 1:1000 dilution. For
staining, Horseradish peroxidase coupled with ECL
detection reagent (GE Healthcare-Amersham, USA)
was used. Anti-β-actin was used as the loading
control antibody. Densitometric analysis was carried
out using Alpha Ease FC software, version 4.0.034
(Alpha Innotech, USA) and results were normalized
to loading control.
Real time PCR
Total RNA from the lumbar spinal cord was extracted
using the standard phenol/chloroform extraction
with TRIzol Reagent (Invitrogen) according to the
manufacturer’s guidelines. Samples were treated
with DNase (Invitrogen) to remove any
contaminating DNA. Total RNA was reverse
transcribed into cDNA using RevertAid First Strand
cDNA Synthesis Kit (Thermo Scientific, USA). All cDNA
samples were stored at -80°C until real-time PCR
(qPCR) was performed.
The primers for TNF-α,IL10, COX-2 and GAPDH were
F: 5 '–GATGGGCTGTACCTTATCTACTCCCAGG-3',R 5'-
CCTTAGGGCAAGGGCTCTTGATGGC; 5' –
TAAGGGTTACTTGGGTTGCCAAGCC-3', reverse 5'–
GCAGCTGTATCCAGAGGGTCTTCAGC - 3'; 5' –
CAGTATCAGAACCGCATTGCCTCTG -3', reverse 5'–
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PhOL Mital Ravalji et al 35 (33-43)
GTGAGCAAGTCCGTGTTCAAGGAGG - 3'; 5' –
CAAGGTCATCCATGACAACTTTGGC - 3', reverse 5'–
CAAGGTCATCCATGACAACTTTGGC - 3', respectively.
Amplification of the cDNA was performed, in a
blinded procedure, using SYBR® Select Master Mix
kit (Life Technologies/ABI) in MicroAmp® 96 well
plate (Life Technologies/ABI) on Step-one Real Time
PCR machine (Applied Biosystems). Each sample
was measured in triplicate. The reactions were
initiated with a hot start at 94ºC for 5 min, followed
by 40 cycles of 5 s at 94ºC (denaturation), 10 sat
55ºC (annealing), and 10 sat 72ºC (extension). Melt
curve analyses were conducted. The comparative
cycle threshold (Ct) method was used for relative
quantification of gene expression. The amount of
mRNA, normalized to the endogenous control
(GAPDH) and relative to a calibrator, was given by 2-
Δ(ΔCt).
Immunohistochemistry
Spinal cord tissues collected after transcardial
perfusion were washed with phosphate buffer and
cryopreserved in sucrose at 4°C, till the tissues
settled at bottom. Frozen tissue was embedded in
OCT (Tissue-Tek, Sakura Finetek, USA) and sections
were taken at 12 µm thickness with cryotome
(Reichert-Jung, Cryocut E Cryostat). Tissues were
immunolabeled using standard immuno-
histochemistry methods for microglial localization
and Iba1 expression using goat polyclonal anti-Iba1
antibody (1:300; Abcam, overnight incubation at
4°C). The ABC-DAB system was used for
immuostaining. From each animal’s spinal cord, four
to five sections within the L4-L5 region were
included in the analysis. The sections were
observed under Leica DM2500 microscope and the
images were captured using EC3 camera utilizing
Leica LAS EZ (V 1.6.0) software. Colour intensity was
quantified using Doc ItLS software (Genei,
Bangalore, India) by an observer unaware of
experimental conditions. The pixel measurement
was used for counting the density-slicing area in the
image of the positive area of the dorsal horn of the
spinal cord. Then, the fold change in the staining
density between NC, CCI-VC and CCI-BIRM was
calculated. The criteria for resting and activated
microglia were as described previously [13]. All
samples from all groups were numbered randomly
and blinded observation was carried out to prevent
bias.
Statistical Analysis
One way ANOVA followed by Tukey’s multiple
comparison test was applied for 50% PWT analysis
and 50% PWL analysis. Unpaired Student’s t-test was
used for analysis of data generated from gene
expression studies. p ≤0.05 was considered
statistically significant. For ease of reading, the basic
statistical values are shown in the text while the
more extensive statistical information can be found
in the figure legends.
Results
The effect of BIRM on CCI -induced changes in
behavioral and neuropathic pain measurements
After CCI surgery, the rats gradually showed the
typical signs of allodynia and hyperalgesia such as toe
closing, foot eversion and paw licking. There was no
significant difference observed in ipsilateral 50% paw
withdrawal threshold (PWT) and paw withdrawal
latency (PWL) among the groups (p>0.05)in animals
subjected to CCI surgery (CCI-VC, CCI-BIRM and CCI-
Gabapentin) after characterization of CCI on day 13
(Table 1 and 2; Figure 1 and 2) .
Mechanical Allodynia
With respect to mechanical allodynia, CCI-vehicle
control group showed significant decrease in 50%
PWT as compared to normal control group
throughout the study duration post CCI induction on
days 13,14,21 and 28 (P ≤0.001). Repeated oral
treatment with BIRM (4 ml/kg) as a single dose daily
for 14 days showed a mean protection of 35% and
38%at 3 h post BIRM administration on day 14 and
day 21, respectively. The increase in 50% PWT was
significantly higher than CCI-vehicle control group
(CCI-VC) on day 14 (p ≤0.05)but not on day 21 as
analysed by One Way ANOVA followed by Tukey’s
multiple comparison test. BIRM showed mean
protection of 70%on day 28. This increase in 50%
PWT was significantly higher than CCI - vehicle
control group (p ≤0.001)as analysed by One Way
ANOVA followed by Tukey’s multiple comparison test
(Table 1; Figure 1). Treatment with Gabapentin at 30
mg/kg showed a mean protection of 82%-83%at all
assessment time i.e., on day 14,21 and 28. The
increase in 50% PWT was significantly higher (P ≤
0.001) than CCI - vehicle control group as analyzed by
One Way ANOVA followed by Tukey’s multiple
comparison test (Table 1; Figure 1).
Thermal hyperalgesia
With respect to thermal hyperalgesia, CCI-Vehicle
control group (CCI-VC) showed significant decrease in
50% PWL throughout the study duration post CCI
induction i.e., on days 14,21 and 28 (p ≤0.001). But
treatment with BIRM (4 ml/kg/day) showed a
significant increase in PWLs at 3h post administration
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on day 14 (p ≤0.05), day 21 (p ≤0.001) and day 28
(p ≤0.001). This increase in PWLs was significantly
higher than CCI - vehicle control group as analysed
by One-way ANOVA followed by Tukey’s multiple
comparison test (Figure 2).
Treatment with Gabapentin showed an average
increase in PWLs at 3hr post administration on day
14,21 and 28. The increase in PWLs was
significantly higher than CCI - vehicle control group
as analysed by One-way ANOVA followed by
Tukey’s multiple comparison test (Table 2; Figure
2).
The effect of BIRM on CCI-induced Iba-1 protein
expression
Quantitative analysis of Western blots showed that
Iba-1 protein level had significantly increased in
lumbar spinal cord tissue on day 28 post CCI surgery
(p<0.001). Significant reduction in Iba-1 protein
level was observed in the CCI group treated with
BIRM (p≤0.01) (Figure 3). These findings in western
blot experiment suggest neuroprotective effect of
repeated oral treatment of BIRM (4 ml/kg/day) on
microglia activation in lumbar spinal cord tissue.
The effect of BIRM on CCI –induced COX-2, TNF-α,
IL-10 expression in spinal cord
Mean fold increase was observed in mRNA levels of
TNF-α(0.022 times) and COX-2 (0.141 times) in
lumbar tissue (L4-L5) in CCI-VC animals as
compared to normal control (NC) animals. Mean
fold change of 0.008 times was observed in mRNA
levels of IL-10 in animals of CCI-VC group. But
treatment with BIRM was able to bring significant
reduction in mean fold change in mRNA levels of
TNF-α(0.007 times)(p ≤0.01) and COX-2 (0.032
times) (p ≤0.001)in lumbar tissue (L4-L5) in CCI-
BIRM animals as compared to CCI-VC group.
Similarly, significant increase in fold change in
mRNA levels of IL-10 (0.029)(p ≤0.01)in CCI-BIRM
group was observed as compared to CCI-VC group.
Results were analysed using Unpaired Student’s t
test (Figure 4).
The effect of BIRM on CCI-induced changes in
spinal microglia cells
Immunohistochemistry was performed using the
Iba-1 antibody which is known to selectively label
activated microglia in nervous tissue. In the normal
control sections, a few Iba-1 positive cells could be
seen (Figure 5A) as compared to higher number of
Iba-1 positive cells in CCI-VC and CCI-BIRM sections
(Figure 5B and 5C). The morphology of microglia
cells in normal control sections exhibited the resting
type shape to some extent, which has small compact
somata bearing long, thin, ramified processes (Figure
5A, arrow marked). Although the thin, ramified
processes are not clearly visible in our sections, soma
diameter is found to be much smaller and compact
as compared to activated microglial cells in CCI-VC
and CCI-BIRM groups. Microglia exhibited an
activated phenotype, showing hypertrophy and
retraction of cytoplasmic processes in the sections of
CCI rats. Compared to normal control rats, a
significant shift from resting to activated morphology
was found in CCI rats. Chronic treatment of BIRM (4
ml/kg) for 14 days was found to reduce the
proportion of the activated phenotype in microglial
cells (Figure 5C). Quantification of Iba-1
immunoreactivity in lumbar dorsal horn shows
significant increase in Iba-1 immunoreactivity in CCI-
VC group as compared to naïve/normal rats
(p≤0.001)but repeated oral treatment of BIRM
significantly inhibits CCI-induced upregulation of Iba-
1 imuunoreactivity as compared to CCI-VC group
(Figure 5D).
Discussion
The present study demonstrates that microglia cells
are the useful tool for evaluating the effects of anti-
neuroinflammatory effects of novel compounds.
Repeated oral administration of BIRM, an aqueous
extract of dried roots of a plant of the species
dulcamara, has significantly inhibited thermal
hyperalgesia and mechanical allodynia in animal
model of CCI-induced neuropathic pain. The
immunohistochemical results have shown the CCI-
induced microglia activation, which is evident from
their morphologies in CCI-VC group. The western blot
results showed increased expression of Iba-1 protein
in lumbar spinal cord in CCI induced neuropathic pain
which supports the immunohistochemical data.
Increased expression of Iba-1 protein under
neuropathic condition indicates activation of
microglia cells in the spinal cord. Repeated
administration of BIRM orally, improved pathological
conditions in animal model of neuropathic pain in the
spinal cord by reducing the expression of Iba-1
protein and the proportion of activated microglia
cells along with significant inhibition of neuropathic
pain symptom, thermal hyperalgesia and mechanical
allodynia. Microglial cells are the resident immune
cells of the central nervous system (CNS). They act as
the main form of active immune defense in the CNS
and upon getting activated following any insult to the
nervous tissue, they become the main source of
inflammatory mediators (e.g.: IL-1β,IL-6, TNF-α,
PGE2, NO, BDNF etc) in the nervous system [14].
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Microglia, once activated, gets engaged in
phagocytosis and also participates in the adaptive
immunity by presenting antigens to T cells. Apart
from their role in inflammatory processes and
regulation of cell survival, they are also capable of
detecting specific aspects of normal and
pathological levels of activity in brain, and its
repercussions. Nerve injury induces extensive
proliferation of spinal microglia and related gene
expression. They become activated and adopt the
immunological functions of the tissue following the
damage [1]. The increased presence of Iba-1
positive cells in L4-L5 region of spinal cord in
present study following induction of neuropathic
pain is in line with the earlier report on the L4-L5
spinal cord dorsal and ventral horn following sciatic
nerve injury [15]. Moreover Patro et al. [16] have
also reported activation of microglia and increased
expression of Iba-1 in the proximity of the sensory
and motor neurons in the L4-L5 spinal cord of the
rats subjected to nerve injury. This data suggests
importance of the role being assayed by Iba-1
protein in regulation of activated microglia
functions. Using Iba-1 as microglia marker, Tawfik et
al. [17] and Romero-Sandoval et al. [18] has also
shown the importance of role being played by
microglia in the maintenance of neuropathic pain
for longer duration. The microglial activation
(presence of Iba-1 positive cells) in the L4-L5 region
of spinal cord following induction of neuropathic
pain through CCI reported in this article supports
the above findings. Repeated administration of
BIRM to CCI rats helps in restoring microglia cells to
its resting stage from the activated stage (Figure
5C). Further, the gene expression analysis of COX-2
and pro-inflammatory cytokines (TNF-α) showed
fold increase in their mRNA levels in lumbar spinal
cord tissue of rats from CCI-induced vehicle control
group as compared to normal control group.
Repeated oral administration of BIRM not only
inhibited the neuropathic pain symptom namely
thermal hyperalgesia and mechanical allodynia but
also prevented CCI-induced changes in spinal cord
and significantly reduced fold increase of
inflammatory mediators like COX-2 and TNF-αin
lumbar spinal cord tissue. At the same time, we
were able to observe fold increase in mRNA levels
of anti-inflammatory cytokine (IL-10)in lumbar
spinal cord tissue of CCI-BIRM treated rats. It is well
documented that COX exists in two isoforms: COX-1
and COX-2. COX-1 is constitutively expressed in
most cells under physiological conditions whereas
COX-2 is highly inducible in response to cytokines,
growth factors, or other inflammatory stimuli and
lasts for several months or even several years [10,
19]. These enzymes catalyse the rate limiting steps of
prostaglandin and thromboxane synthesis.
Prostaglandins play a crucial role in nociceptive
transmission at peripheral sites and in the spinal cord
[20,21]. The present gene expression studies show
the mRNA levels of COX-2 in lumbar spinal cord of
naïve rats (Figure 4). COX-2, being an inducible
enzyme, increases in the peripheral and central
nervous system post injury or inflammation [22,23]
and plays an important role in neuropathology. Jean
et al. [24] observed overexpression of COX-2 in
injured nerve in rats following CCI, partial sciatic
nerve ligation, spinal nerve ligation and complete
sciatic nerve transaction intervention. Supporting this
observation, the present gene expression study
shows a significant increase in COX-2 mRNA levels in
the lumbar spinal cord in CCI rats as compared to
normal/naïve rats (Figure 4). Matsunaga et al. [25]
showed that inhibition of COX-2 by selective
inhibitors attenuates hyperalgesia in neuropathic
rats. This increase in COX-2 mRNA levels was
inhibited by repeated oral treatment of BIRM in CCI-
BIRM treated rats. These results suggest that BIRM
produces an analgesic effect on neuropathy via
inhibition of the expression of COX-2 mRNA levels in
the spinal cord. The current results support the
earlier findings reported by Jaggi et al. [10]
demonstrating inhibitory effect of mother tincture
Solanum dulcamara on PGE2production via COX-1
and COX-2 in vitro.In addition, central neuroimmune
activation and neuro-inflammation have also been
postulated to mediate and/or modulate the
pathogenesis of persistent pain states. Pro-
inflammatory cytokines (IL-1β,IL-6 and TNF-α),
signaling proteins are uniquely powerful and have
been associated with cell proliferation,
differentiation and changes in gene expression and
synthesis of matrix proteins important to cell growth
and tissue repair [26]. They induce a long term
alteration of synaptic transmission in the CNS and
play a critical role in the development and
maintenance of neuropathic pain [27,28], but on the
other hand they are also essential in fighting
infection and responding to injuries. Each of these
pro-inflammatory cytokines has been observed in
spinal cord under pathological conditions implicating
its role in pain facilitation. These cytokines activate
neurons as wells as glia via specific receptors. In the
CNS, the major contributors of cytokine release are
glia. Microglia can produce cytokines on activation
[29]. Nerve injury or peripheral inflammation has
been reported to activate glial cells and increase the
pro-inflammatory cytokine levels in the CNS [30].
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Also as per the previous findings, TNF-α,IL-1 and
/or IL-6 mRNA expression is elevated in spinal cord
in response to peripheral nerve injury [31], spinal
nerve injury [32], each of which elevates pain
responses (hyperalgesia and allodynia). In line with
this, the present study showed fold increase in
mRNA levels of TNF- αin the lumbar spinal cord of
rats following the sciatic nerve ligation (CCI) (Figure
4). Repeated oral administration of BIRM to CCI-rats
lowered the fold increase in mRNA levels of pro-
inflammatory cytokine (TNF-α) (Figure 4) and these
could be due to its direct interaction with immune
cells of the CNS. There are reports showing increase
in TNF-α,IL-1 and/or IL-6 protein levels in spinal
cord following peripheral nerve injury [32,33].
IL-10, being a suppressor of macrophages, is
considered as an anti-inflammatory cytokine. It
potently down-regulates production and release of
pro-inflammatory cytokines like TNF-α,IL-1βand IL-
6 [34]. Although the precise functions of IL-10 in the
CNS require further clarification, it is well known as
an important negative regulator of pro-
inflammatory gene expression. It can down-regulate
the expression of receptors for pro-inflammatory
cytokines [35,36]. In our present study we
observed significant fold increase in IL-10 mRNA
levels in the lumbar spinal cord of CCI-rats treated
with BIRM as compared to CCI-vehicle treated rats
(Figure 4). Ledeboer et al. [37] have reported that
IL-10, when injected in a region of the spinal cord
where activated glial cells were present, drastically
reduced the pain symptoms in animal models of
chronic pain. The latter record consolidates our
current finding of BIRM induced heightened
expression of IL-10 with concomitant reduction in
the expression of TNF-α. This together with the
observed reduction in the expression of COX-2 and
the attended decline in prostanoid synthesis explain
the reasons for the effective amelioration of
neuropathic pain observed in BIRM treated rats.
In summary, our study with BIRM shows inhibition
of microglia activation in the CNS and
downregulates pro-inflammatory cytokines and
COX-2. As demonstrated in this study, BIRM
attenuates the development of hyperalgesia and
allodynia in the rat model of neuropathic pain.
Overall, this study not only demonstrates the
effectiveness of BIRM in improving pathological
conditions of nerve injury induced neuropathic pain
but also showed the important role played by
microglia in regulating the induction of a chronic
pain state induced by peripheral nerve ligation.
Based on the results obtained using BIRM as a
therapeutic agent in neuropathic pain model at
preclinical stage, BIRM has potential to improve pain
condition or reduce the disease progression as a
standalone therapeutic or adjuvant to standard
therapy.
Conflict of Interest
There is no conflict of interest.
Acknowledgements
We are thankful to GVK Biosciences Pvt. Ltd. for
providing infrastructure assistance in carrying out
these studies. We are also thankful to Dr. Amit
Sharma for his assistance in surgical procedures and
to Dr. Chetan Sharma and Dr. Mukesh Gandhari for
technical assistance. PB is grateful to the Council of
Scientific & Industrial Research, New Delhi, India for
financial assistance as fellowship.
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ISSN: 1827-8620
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ISSN: 1827-8620
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Group 50% PWT of Ipsilateral Paws on Days
013 14 21 28
Normal Control 14.25±0.49 14.62±0.38 14.42±0.58 14.62±0.38 14.25±0.49
CCI-Vehicle control 14.62±0.38 3.00±0.54 3.11±0.53 4.30±1.28 2.63±0.28
CCI-BIRM 14.62±0.38 3.02±0.20 7.31±1.46* 8.34±1.29 11.31±1.24***
CCI-Gabapentin 14.62±0.38 3.11±0.28 12.98±0.90*** 13.03±1.08*** 12.84±1.02***
Table 1. Effect of BIRM on paw withdrawal threshold in CCI-induced neuropathic pain in rats
Values represented as Mean±SEM. PWT = paw withdrawal threshold, *p ≤ 0.05 , ***p ≤ 0.001 as
compared to CCI-vehicle control group. Data analyzed by one-way ANOVA followed by Tukey’s multiple
comparison test.
Group
50% PWL of Ipsilateral Paws on Days
013 14 21 28
Normal Control 19.70±0.20 19.29±0.36 19.35±0.22 19.62±0.27 19.75±0.17
CCI-Vehicle control 19.71±0.18 7.38±0.47 8.60±0.23 7.61±0.38 7.31±0.58
CCI-BIRM 19.84±0.11 7.84±0.40 12.27±1.37* 13.18±1.32*** 15.47±0.55***
CCI-Gabapentin 19.63±0.26 7.55±0.47 16.23±1.05*** 16.13±0.17*** 16.47±0.35***
Table 2. Effect of BIRM on paw withdrawal latency in CCI-induced neuropathic pain in rats
Values represented as Mean±SEM.sed by PWL= paw withdrawal latency, *p ≤0.05, ***p ≤0.001 as
compared to CCI-vehicle control group. Data analyzed by one-way ANOVA followed by Tukey’s multiple
comparison test.
Figure 1. Effect of BIRM treatment on
mechanical sensitivity in rats after CCI on the
sciatic nerve. Response to tactile mechanical
stimulus was measured in animals prior to
surgery (day 0), prior to treatment (Day 13),
and at 3 h post treatment on day 14,21 and
28. Data presented are mean ±SEM of
median force (g) required to induce paw
withdrawal in animals.
*p ≤0.05, ***p ≤0.001, as compared to CCI-
vehicle control group. Significant changes
were observed in CCI-vehicle control group
as compared to Normal Control group (###p≤
0.001). Data analyzed by one-way ANOVA
followed by Tukey’s multiple comparison
test.
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Figure 2. Effect of BIRM treatment on thermal
hyperalgesia in rats after CCI on the sciatic nerve.
Response to thermal stimulus was measured in
animals prior to surgery (day 0), prior to
treatment (Day 13), and at 3 h post treatment on
day 14,21 and 28. Data presented are mean ±
SEM of time (s) taken to respond (PWL) to
thermal hyperalgesic stimulus.
*p ≤0.05, ***p ≤0.001, as compared to CCI-
vehicle control group. Significant changes were
observed in CCI-vehicle control group as
compared to Normal Control group (###p ≤
0.001). Data was analyzed by one-way ANOVA
followed by Tukey’s multiple comparison test.
Figure 3. Suppression of upregulated Iba-1 in the lumbar spinal cord tissue in CCI animals post BIRM treatment. A
significant increase in expression of Iba-1 protein was observed post CCI surgery. BIRM treatment shows visibly
significant reduction in upregulation of Iba-1 expression (Figure 3A). Figure 3B depicts relative intensities of bands.
Data presented are mean±SEM of relative density Iba-1/β-actin. **p ≤0.01 as compared to CCI-VC, ###p ≤0.001
as compared to Normal control group. Data analysed by One-way ANOVA followed by Tukey’s multiple
comparison test.
Figure 4. Mean Fold change in mRNA
levels of COX-2, TNF-α and IL-10 as
compared to Normal control rats.
Tissue (dorsal lumbar spinal cord)
collected 2 wks after administration of
BIRM (4 ml/kg, p.o.) or vehicle in CCI
rats.
**p ≤ 0.01, ***p ≤ 0.001 as compared
to CCI-VC. Data analysed using
unpaired Student’s t test.
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Figure 5. Immunostaining images of microglia cells labelled with Iba-1 (activated microglia cell marker) for the
lumbar spinal cord sections.
Figures 5A, 5B and 5C respectively show Iba-1 immunoreactivity in the lumbar spinal cord of naive/control rats,
CCI-VC rats and CCI-BIRM rats on day 28 post CCI surgery. Basal levels of Iba-1 labelled microglia cells were
observed in naive animal with resting morphology (A, arrow marked). Activated phenotype with marked cellular
hypertrophy and retraction of processes as compared with control animals was observed in CCI rats (B). BIRM (4
ml/kg, p.o.) inhibits CCI-induced microglial activation to a large extent and reduces the activated phenotype of
microglia cells and shift towards the resting stage was observed (C). Scale bar represents 100μm.
Figure 5D shows Quantification of Iba-1 immunoreactivity indicating that BIRM significantly inhibited CCI-induced
microglial activation in the dorsal horn of the spinal cord.
Data was analysed using one way ANOVA, followed by Tukey’s multiple comparison test. ###p ≤0.001 as compared
to NC, **p ≤0.01 as compared to CCI-VC.