Neurochem Res (2014) 39:21362142

DOI 10.1007/s11064-014-1407-y

ORIGINAL PAPER

Attenuation of Neuropathic Pain by Saikosaponin

a in a Rat Model of Chronic Constriction Injury
Xin Zhou Hong Cheng Dedong Xu
Qing Yin Lei Cheng Lei Wang Shasha Song

Mengyuan Zhang

Received: 14 June 2014 / Revised: 29 July 2014 / Accepted: 31 July 2014 / Published online: 9 August 2014
Springer Science+Business Media New York 2014

Abstract Despite immense advances in the treatment
strategies, the effective treatment of patients suffering from
neuropathic pain remains challenging. Saikosaponin a pos-
sesses anti-inflammatory activity. However, the role of sai-
kosaponin a in neuropathic pain is still unclear. Therefore,
the objective of this study was to investigate the effects of
saikosaponin a on neuropathic pain. Neuropathic pain was
induced by chronic constriction injury (CCI) of the sciatic
nerve in rats. After CCI, rats were administered saikosaponin
a (6.25, 12.50 and 25.00 mg/kg intraperitoneal, once daily)
for 14 days. Mechanical withdrawal threshold and thermal
Xin Zhou and Hong Cheng have contributed equally to this work.
withdrawal latency were assessed before surgery and on days
1, 3, 7, and 14 after CCI. Our results showed that CCI sig-
nificantly decreased mechanical withdrawal threshold and
thermal withdrawal latency on days 1, 3, 7 and 14, as com-
pared with sham groups, however, saikosaponin a reversed
this effects. In addition, saikosaponin a inhibited CCI-
induced the levels of TNF-a, IL-1b, IL-2 in spinal cord.
Western blot analysis demonstrated that saikosaponin a
reduced the elevated expression of p-p38 mitogen-activated
protein kinase (MAPK) and NF-jB in the spinal cord
induced by CCI. These results suggest that saikosaponin a
could effectively attenuate neuropathic pain in CCI rats by
inhibiting the activation of p38 MAPK and NF-jB signaling
pathways in spinal cord.

X. Zhou  M. Zhang (&) Keywords Saikosaponin a  Neuropathic pain 

Department of Anesthesiology, Shandong Provincial Hospital,
Shandong University, 324#, Jing Wu Wei Qi Road,
Jinan 250021, Shandong, China
e-mail: mengyuan_zhangjn@126.com
H. Cheng  D. Xu
Department of Anesthesiology, Jinan Central Hospital,
Shandong University, Jinan 250013, Shandong, China
Q. Yin
Department of Operation, Jinan Central Hospital, Shandong
University, Jinan 250013, Shandong, China
L. Cheng
Medical Department, Peoples Hospital,
Lin Qing City, Linqing 252600, Shandong, China
L. Wang
Department of Anesthesiology, The Peoples Liberation Army
456 Hospital, Jinan 250031, Shandong, China
S. Song
Department of Pathology, Jinan Central Hospital, Shandong
University, Jinan 250013, Shandong, China
Pro-inflammatory cytokines
Introduction
Neuropathic pain is defined as pain that originates from a
lesion or disease that affects the somatosensory pathways
within the peripheral or central nervous system [1]. It
affects approximately 7 % of the European population; and
about a fifth of people who report chronic pain are thought
to have predominantly neuropathic pain [2]. Despite recent
advances made in treatment of neuropathic pain, current
pharmaceutical treatment is frequently unsatisfactory.
Therefore, there is a need to develop new strategies for the
treatment of neuropathic pain with acceptable adverse side
effects.
Several lines of evidence have shown that natural pro-
ducts, especially medicinal plants, may represent an ideal
source to develop safe and effective agents for management

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Neurochem Res (2014) 39:21362142
of neuropathic pain. Kim et al. [3] reported that Ginkgo bi-
loba extract EGb 761 could significantly reduce the paw
withdrawal thresholds to mechanical stimuli and withdrawal
frequencies to cold stimuli. Sinomenine is an alkaloid orig-
inally isolated from the root of the plant Sinomenium acu-
tum, which effectively alleviated mechanical and cold
allodynia in rats and mice after injury to peripheral nerve or
spinal cord [4]. Intraperitoneal administration of curcumin
was shown to efficiently alleviate the development of
chronic neuropathic pain in rats with peripheral nerve injury
[5].
Radix Bupleuri, with a Chinese name Chaihu, which
belongs to the family Umbelliferae, is a kind of herbal plant
distributed mainly in Hubei and Sichuan Provinces of China.
It has been widely used the treatment of common cold with
fever, hepatitis, kidney syndrome, inflammatory diseases,
menoxenia, etc. [6]. Saikosaponins is the major chemical
constituent isolated from RB, which have been demonstrated
to possess anti-inflammatory, immune-regulating, antibac-
terial and antiviral activities [79]. Among saikosaponins,
saikosaponin a is known as the major active constituents
which had anti-inflammatory action [10]. However, the role
of saikosaponin a in neuropathic pain is still unclear.
In this study, we investigated the effects of saikosaponin
a on neuropathic pain induced by mouse sciatic nerve
chronic constriction injury (CCI). In addition, we investi-
gated the molecular mechanisms involved in the saikosa-
ponin a-induced suppression of neuropathic pain.
Materials and Methods
Animals
Adult male SpragueDawley rats, weighting 200220 g,
were purchased from the Laboratory Animal Center. The
animals were housed in a room maintained at 22 1 C
with an alternating 12-h lightdark cycle, and provided
food and water ad libitum. Animal experiments conformed
to the guidelines issued by the Shandong Provincial Hos-
pital of Shandong University. The present study was per-
formed with approval from the Animal Ethics Committee
of Shandong Provincial Hospital, Shandong University.
Drug Treatment
Saikosaponin a was obtained from Sigma (St. Louis, MO,
USA). Rats were randomly divided into 5 groups with 10
rats in each group: sham group, CCI group, saikosaponin a
(6.25 mg/kg) group, saikosaponin a (12.50 mg/kg) group
and saikosaponin a (25.00 mg/kg) group. Rats were intra-
peritoneally administered saikosaponin a in doses of 6.25,
12.50 and 25.00 mg/kg, once daily, respectively, and sham
2137
group was administered 0.9 % saline, for 2 weeks follow-
ing the induction of neuropathic pain.
Induction of Neuropathic Pain
The CCI of sciatic nerve was used as a model for the
induction of neuropathic pain [11]. In brief, animals were
anesthetized with sodium pentobarbital (40 mg/kg, i.p.).
The sciatic nerve was exposed and loosely ligated with 4-0
chromic gut thread at 4 sites with an interval of 1 mm.
Meanwhile, a sham surgery was performed with the sciatic
nerve exposed but not ligated.
Evaluation of Thermal Hyperalgesia and Mechanical
Allodynia
Rats were placed in an inverted clear plexiglass cage
(23 9 18 9 13 cm) on a piece of 3-mm-thick glass plate
and allowed to acclimatize for 30 min before testing.
Mechanical hyperalgesia was evaluated by von Frey fila-
ments as described previously [12]. Animals were placed
on an elevated wire grid and the plantar surface of the hind
paw was stimulated with a series of von Frey filaments.
Quick withdraw or licking of the paw in response to the
stimulus was considered a positive response.
Heat hypersensitivity was tested using a plantar test (7370,
UgoBasile, Comeria, Italy) according to the method described
by Hargreaves et al. [13]. After acclimation, the heat source
was positioned under the glass floor directly beneath the hind
paw. The intensity of the thermal stimulus was adjusted to
achieve an average baseline paw withdrawal latency of
approximately 911 s. A digital timer automatically recorded
the duration between the start of stimuli and the paw with-
drawal thermal latency (PWTL). Each paw was measured
alternatively after more than 5 min. The cut-off was set at 20 s
to avoid tissue damage. Tests were performed 1 day before
CCI surgery, and 1, 3, 7 and 14 days after CCI surgery.
RT-PCR
Total RNA from spinal cord tissues was extracted using the
RNAiso plus kit (Takara Biotechnology, Dalian, China)
according to the manufacturers instructions. Then, the iso-
lated RNA was reverse-transcribed to synthesize the first
strand cDNA with the oligo (dT)18 primer using the cDNA
synthesis kit (Takara Biotechnology, Dalian, China), the
obtained cDNA was used as a template to perform PCR
amplification using SYBR Premix Ex TaqTM II kit (Takara
Biotechnology, Dalian, China). The specific primers for
NF-jB p65 were sense, 50 -GTGCAGAAAGAAGACATTG
AGGTG-30 and antisense, 50 -AGGCTAGGGTCAGCGTAT
GG-30 ; and for b-actin were sense, 50 -AAATCG TGCGTG
ACATCAAAGA-30 and antisense, 50 -GGCCATCTCCTGC
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TCGAA-30 . Each 20 ll reaction system comprised 2 ll of
cDNA, 10 ll SYBR Premix Ex Taq II, 10 lmol/l of both
sense and antisense primers. For normalization, b-actin was
used to normalize mRNA. Each experiment contained at least
three replicates, and the results were calculated according to
the method 2-DDCt.
Western Blot
The proteins were extracted from spinal cord tissues using
RIPA lysis buffer (Beyotime, Nantong, China) according to
the operating instructions. The protein concentration in the
lysates was evaluated using a BCA protein assay kit (Beyo-
time, Nantong, China). Proteins were separated by SDS-
PAGE, transferred to a nitrocellulose membrane (Millipore,
Boston, MA, USA). Membranes were blocked with 10 % fat
milk in TBS containing 0.1 % Tween 20 and probed with anti-
NF-jB p65, anti-p-p38, anti-p38 or b-actin) (Abcam, Cam-
bridge, MA) overnight at 4 C. Then, the blots were washed
and incubated with horseradish peroxidase-conjugated sec-
ondary antibody. Protein-antibody complexes were visualized
using the enhanced chemiluminescence western blotting
detection system according to the manufacturers protocol
(GE, Orsay, France). b-Actin was used as internal control. At
least three independent experiments were performed.
Enzyme Linked Immunosorbent Assay (ELISA)
Rats were anesthetized with sodium pentobarbital 12 h
after the last treatment. Lumbar segments of the spinal cord
were removed and homogenized in lysis buffer. After
centrifugation, the supernatants were measured by the Rat
TNF-a ELISA kit, IL-1b and IL-2 (Invitrogen, Carlsbad,
CA, USA) according to the manufacturers instructions. All
samples were processed in triplicate and data were
obtained from three independent experiments.
Statistical Analysis
All data were presented as the mean SEM. The behav-
ioral data were analyzed by two-way ANOVA followed by
the Turkeys test for post hoc analysis. The data from the
biochemical tests and western blot were analyzed with one-
way ANOVA followed by LSD post hoc test. Values of
P \ 0.05 were considered as statistically significant.
Results
The Effect of Saikosaponin a on Thermal Heperalgesia
and Mechanical Allodynia
After CCI, rats were administered saikosaponin a (6.25,
12.50, and 25.00 mg/kg intraperitoneal, once daily) for a
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Neurochem Res (2014) 39:21362142
period of 14 days. Mechanical withdrawal threshold and
thermal withdrawal latency were assessed before surgery
and on days 1, 3, 7, and 14 after CCI. As shown in Fig. 1,
saikosaponin a reversed CCI-induced mechanical allodynia
and thermal hyperalgesia [F(4,116) = 32.315, P \ 0.001,
and F(4,116) = 39.976, P \ 0.001]. One day after injury,
thermal withdrawal latency and withdrawal threshold were
significantly decreased in CCI group and saikosaponin
a-treated groups, as compared with the sham group; 3 days
after injury, thermal hyperalgesia and mechanical allodynia
were still present in CCI group, whereas saikosaponin a
(12.50 and 25.00 mg/kg) reversed CCI-induced mechanical
allodynia and thermal hyperalgesia and 6.25 mg/kg saiko-
saponin a was ineffective. In addition, saikosaponin a
administration in a dose of 25.00 mg/kg for 14 days resulted
in the most pain relief, as compared to the CCI group.
Effects of Saikosaponin a on Pro-inflammatory
Cytokines in Spinal Cord
The dysregulation of cytokines is a feature in the development
of neuropathic pain. Therefore, we investigated the effects of
saikosaponin a on pro-inflammatory cytokines in spinal cord.
As shown in Fig. 2, there was significant increases in the
proteins levels of TNF-a, IL-1b and IL-2 in spinal cord of CCI
rats, as compared with the sham group. However, saikosa-
ponin a reversed CCI-increased the levels of TNF-a, IL-1b
and IL-2 [F(4,20) = 33.199, P \ 0.001; F(4,20) = 6.351,
P = 0.002; F(4,20) = 34.070, P \ 0.001]. Taken together,
these results suggest that saikosaponin a attenuated the
expression of TNF-a, IL-1b and IL-2 in spinal cord of CCI
rats.
Effects of Saikosaponin a on p-p38 MAPK in Spinal
Cord After CCI
Previous studies demonstrated that the activation of p38 in the
nociceptive pathway contributes to the development of
inflammatory and nerve injury induced neuropathic pain [14,
15]. Therefore, we investigated the effects of saikosaponin a
on phosphorylation of p38 mitogen-activated protein kinase
(p-p38 MAPK) in spinal cord. As shown in Fig. 3, the
expression level of p-p38 MAPK was significantly increased
by CCI, as compared with the sham group. However, saiko-
saponin a obviously inhibited this effect [F(4,15) = 28.990,
P \ 0.001]. These result demonstrated that saikosaponin a
showed inhibitory effect on p38MAPK pathway.
Saikosaponin a Reduces the Expression of NF-jB p65
in Spinal Cord After CCI
NF-jB is a key transcription factor complex that controls
the expressions of pro-inflammatory and pain mediators
Neurochem Res (2014) 39:21362142
Fig. 1 Saikosaponin a
attenuates CCI-induced
neuropathic pain. The hind paw
withdrawal threshold (a), and
the hind paw withdrawal latency
(b), decreased progressively in
the CCI group. Treatment with
saikosaponin a obviously
inhibited the development of
CCI-induced thermal
hyperalgesia and mechanical
allodynia. n = 5, #P \ 0.05
versus sham group; *P \ 0.05
versus CCI group
[16]. To investigate the mechanism of saikosaponin a in
CCI-induced neuropathic pain, we examined the expres-
sion of NF-jB p65 associated with NF-jB signaling
pathway in spinal cord of CCI rats. As shown in Fig. 4,
compared with the sham group, CCI group showed sig-
nificantly higher levels of NF-jB p65 mRNA and protein.
Whereas, saikosaponin a markedly decreased the expres-
sion of NF-jB p65 mRNA in spinal cord of CCI rats,
exhibiting a dose-dependent manner [F(4,20) = 33.018,
P \ 0.001]. Consistent with the results of RT-PCR, the
results of western blot also showed that saikosaponin a
decreased the expression of NF-jB p65 protein in spinal
cord of CCI rats [F(4,20) = 31.491, P \ 0.001]. The
present results clearly indicated that saikosaponin a showed
inhibitory effect on NF-jB pathway.
Discussion
The main findings of our study is that saikosaponin a
reversed CCI-decreased mechanical withdrawal threshold
and thermal withdrawal latency; saikosaponin a inhibited
CCI-induced the levels of pro-inflammatory cytokines
TNF-a, IL-1b and IL-2 and in spinal cord. Furthermore,
2139
saikosaponin a reduced the elevated expression of p-p38
MAPK and NF-jB in spinal cord induced by CCI.
Several animal models have been developed to study
neuropathic pain mechanisms. Of these, the CCI of the
sciatic nerve is probably the most frequently used model
for the study of neuropathic pain, due to its similarities
with human behavioural responses [17]. In this study, we
used CCI model to investigate the effects of saikosaponin a
on neuropathic pain, we found that CCI produced obvious
mechanical allodynia and thermal hyperalgesia, which
suggest that the model was successful. In addition, we
found that saikosaponin a attenuated mechanical allodynia
and thermal hyperalgesia for 14 days, suggesting the pos-
sible of therapeutic efficacy of saikosaponin a.
It has been reported that saikosaponin a and its epimer
saikosaponin d inhibit the production of the pro-inflam-
matory cytokines nitric oxide, prostaglandin E2 (PGE2)
and TNF-a in lipopolysaccharide-induced macrophages,
and these two compounds exert their anti-inflammatory
activity by down-regulation of inducible nitric-oxide
synthase (iNOS) and cyclooxygenase-2 (COX-2) expres-
sion via blocking NF-jB activation [8]. In addition, pro-
inflammatory cytokines have been involved in demyelin-
ation and degeneration of peripheral nerves, increase in
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b Fig. 2 Sikosaponin a attenuated the expression of TNF-a, IL-1b and

IL-2 in the spinal cord of CCI rats. Rats were anesthetized with
sodium pentobarbital 12 h after the last treatment. Lumbar segments
of the spinal cord were removed and homogenized in lysis buffer.
After centrifugation, the supernatants were measured. CCI signifi-
cantly increased the expression of TNF-a, IL-1b and IL-2 in the
spinal cord, whereas administration of skosaponin a inhibited this
effect. Data shown as mean SEM. n = 5, *P \ 0.05 versus sham
group; #P \ 0.05 versus CCI group

sensory afferent excitability, and induction of neuropathic

pain [18]. In animal models, administration of cytokine
inhibitors before nerve injury reduces neuropathology and
pain-related behaviors. For example, intrathecal injection
of IL-1b induces mechanical allodynia, and IL-1 receptor
antagonist can inhibit hyperalgesic responses to IL-1b
[19]. In this study, we found that saikosaponin a signifi-
cantly suppressed the over-expression of spinal TNF-a,
IL-1b and IL-2 in CCI model of rats in a dose-dependent
manner. We conclude that the analgesic effect of saiko-
saponin a might be mediated, at least partly, through the
prevention of TNF-a, IL-1b and IL-2 products.
It is well known that the activation of NF-jB pathway
is a key event for the transmission and processing of
nociceptive information [20]. A variety of inflammatory
mediators, such as TNF-a, IL-1b, IL-2, NO and TGF-1b,
which can activate NF-jB, are implicated in the regula-
tion of the neuropathic pain [21]. Pretreatment with
NF-jB inhibitor (PDTC) improved mechanical allodynia
and down-regulated the over-expression of TNF-a and
tumor necrosis factor receptor 1 (TNFR1) induced by
peri-sciatic administration of TNF [22]. In this study, we
found that saikosaponin a reduced CCI-elevated expres-
sion of NF-jB in spinal cord. Furthermore, it has been
reported that phosphorylated p38 (p-p38) MAPK, the
active form of p38 MAPK, is induced in spinal cord
microglia after peripheral nerve injury and that adminis-
tration of a p38 MAPK inhibitor into the spinal cord
suppresses the development of the nerve injury-induced
tactile allodynia [23, 24]. Consistent with these studies,
we found that CCI could increase the expression level of
p-p38 MAPK in spinal cord. In addition, the present study
showed that saikosaponin a reduced the elevated expres-
sion of p-p38 MAPK in the spinal cord induced by CCI.
Therefore, it is reasonable to assume that saikosaponin a
could attenuate neuropathic pain in CCI rats by inhibiting
the activation of p38MAPK and NF-jB signaling path-
ways in spinal cord.

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Neurochem Res (2014) 39:21362142
Fig. 3 Effects of saikosaponin a on p-p38 MAPK in spinal cord after
CCI. Phosphorylation of p38 in spinal cord 14 days after injection of
skosaponin a. Representative western blots for p-p38, total p38 or
b-actin for spinal cord samples. These data represents mean SEM,
n = 4 mice per group. *P \ 0.05 versus sham group; #P \ 0.05
versus CCI group
In conclusion, the present study suggests that the
potential use of saikosaponin a in the treatment of neuro-
pathic pain, which merits further clinical investigation.
2141
Fig. 4 Saikosaponin a reduces the expression of NF-jB p65
following CCI. Rats were anesthetized with sodium pentobarbital
12 h after the last treatment. The proteins were extracted from spinal
cord tissues using RIPA lysis buffer. a RT-PCR quantitation of
NF-jB p65 expression mRNA in spinal cord. CCI significantly
increased the expression of NF-jB p65 mRNA in the spinal cord,
whereas saikosaponin a injection inhibited the upregulation of NF-jB
p65 mRNA. b The protein level of NF-jB in spinal cord was
determined by western blot analysis, b-actin was used as a loading
control. All experiments were repeated at least three times. Data
shown as mean SEM. n = 5, #P \ 0.05 versus sham group;
*P \ 0.05 versus CCI group
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