Inhibition of the Cyclic Adenosine Monophosphate Pathway Attenuates Neuropathic Pain and Reduces Phosphorylation of Cyclic Adenosine Monophosphate Response Element-Binding in the Spinal Cord After Partial Sciatic Nerve Ligation in Rats

Article (PDF Available)inAnesthesia and analgesia 105(6):1830-7, table of contents · January 2008with168 Reads
DOI: 10.1213/01.ane.0000287652.42309.5c · Source: PubMed
Abstract
Recent reports have identified a role for cyclic adenosine monophosphate (cAMP) transduction in nociceptive processing. Spinal activation of the cAMP induced gene transcription through the activation of protein kinase A and cAMP response element-binding protein (CREB). Intrathecal injection of protein kinase A inhibitor reversed the mechanical hyperalgesia, whereas injection of CREB antisense attenuated tactile allodynia caused by partial sciatic nerve ligation (PSNL) in rats. In the present study, we aimed to assess the effects of spinal cAMP transduction on the nociceptive processing in a chronic neuropathic pain model. PSNL was performed in male Sprague-Dawley rats 1 wk after intrathecal catheterization. Nociception to mechanical and thermal stimuli was assessed at the hindpaw 2 h, 3, 7, and 14 days after PSNL. The effects of adenylate cyclase inhibitor, SQ22536 (0.7 mumol, intrathecal) on these nociceptions were evaluated. Changes in the expression and immunoreactivity of CREB and its phosphorylated proteins (CREB-IR and pCREB-IR) in the dorsal horn of the spinal cord were also measured. The expression of CREB-IR and pCREB-IR proteins was shown to increase for 2 wk after PSNL. The increase in pCREB was partially reversed by the blockade of the cAMP pathway in the early 3 days, with a parallel increase in mechanical and thermal withdrawal thresholds. These results revealed the possible contribution of an increase in pCREB to the PSNL-induced tactile allodynia and thermal hyperalgesia. Modulation of the cAMP pathway may be clinically relevant if early intervention can be achieved in patients with chronic neuropathic pain.

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Available from: Jiin-Tarng Liou, Jun 09, 2014
Inhibition of the Cyclic Adenosine Monophosphate
Pathway Attenuates Neuropathic Pain and Reduces
Phosphorylation of Cyclic Adenosine Monophosphate
Response Element-Binding in the Spinal Cord After
Partial Sciatic Nerve Ligation in Rats
Jiin-Tarng Liou, MD*†
Fu-Chao Liu, MD*†
Shi-Tai Hsin, MD*
Ching-Yue Yang, MD*
Ping-Wing Lui, MD, PhD‡
BACKGROUND: Recent reports have identified a role for cyclic adenosine monophos-
phate (cAMP) transduction in nociceptive processing. Spinal activation of the
cAMP induced gene transcription through the activation of protein kinase A and
cAMP response element-binding protein (CREB). Intrathecal injection of protein
kinase A inhibitor reversed the mechanical hyperalgesia, whereas injection of
CREB antisense attenuated tactile allodynia caused by partial sciatic nerve ligation
(PSNL) in rats. In the present study, we aimed to assess the effects of spinal cAMP
transduction on the nociceptive processing in a chronic neuropathic pain model.
METHODS: PSNL was performed in male Sprague-Dawley rats 1 wk after intrathecal
catheterization. Nociception to mechanical and thermal stimuli was assessed at the
hindpaw 2 h, 3, 7, and 14 days after PSNL. The effects of adenylate cyclase
inhibitor, SQ22536 (0.7
mol, intrathecal) on these nociceptions were evaluated.
Changes in the expression and immunoreactivity of CREB and its phosphorylated
proteins (CREB-IR and pCREB-IR) in the dorsal horn of the spinal cord were also
measured.
RESULTS: The expression of CREB-IR and pCREB-IR proteins was shown to increase
for 2 wk after PSNL. The increase in pCREB was partially reversed by the blockade
of the cAMP pathway in the early 3 days, with a parallel increase in mechanical and
thermal withdrawal thresholds.
CONCLUSION: These results revealed the possible contribution of an increase in
pCREB to the PSNL-induced tactile allodynia and thermal hyperalgesia. Modula-
tion of the cAMP pathway may be clinically relevant if early intervention can be
achieved in patients with chronic neuropathic pain.
(Anesth Analg 2007;105:1830 –7)
Neuropathic pain resulting from nerve injury due
to trauma or disease is one of the most difficult
challenges in pain management because of its varied
mechanisms. Several reports have identified the roles
in the cascade of cyclic adenosine monophosphate
(cAMP) transduction involving nociceptive process-
ing. Gene transcription was induced through the
activation of protein kinase A (PKA) with the subse-
quent phosphorylation of the transcription factor, i.e.,
cAMP response element-binding protein (CREB) (1).
In addition, several lines of evidence showed an
increase in the phosphorylation of CREB in the super-
ficial dorsal horn of neurons after partial sciatic nerve
ligation (PSNL) (2). Peripheral activation of the ade-
nylate cyclase-cAMP pathway was involved in the
mediation of hyperalgesia in rats (3). Although an
increase in cAMP was crucial in producing mechanical
hyperalgesia, the PKA and CREB activities were also
important in the maintenance of inflammatory pain
(4). Injection of PKA inhibitor into the rodent spinal
cord reversed mechanical hyperalgesia in response to
intradermal injection of capsaicin (5). Furthermore,
phosphorylation of CREB secondary to mechanical
hyperalgesia was time-dependently reversed by
blockade of the cAMP pathway after repeated intra-
muscular (IM) injections of acid (3). There is also some
evidence that intrathecal (i.t.) injection of CREB anti-
sense oligonucleotide attenuated PSNL-induced tactile
From the *Department of Anesthesiology, Chang Gung Memorial
Hospital; †Graduate Institute of Clinical Medical Sciences, Chang
Gung University, Taoyuan; and ‡Suao and Yuanshan Veterans Hos-
pital, Yilan, National Yang-Ming University, Taipei, Taiwan.
Accepted for publication August 20, 2007.
Supported by a research grant (CMRPG 33012 and partial of
CMRPG 34028) to J.T. Liou provided by Chang Gung Memorial
Hospital and Graduate Institute of Clinical Medical Sciences, Chang
Gung University, Taiwan.
Address correspondence and reprint requests to Ping-Wing Lui,
MD, PhD, Suao and Yuanshan Veterans Hospital, No. 386, Rong-
guang Rd., Yuanshan Township, Yilan County 264, Taiwan. Ad-
dress e-mail to pwlui@mail.ysvh.gov.tw.
Copyright © 2007 International Anesthesia Research Society
DOI: 10.1213/01.ane.0000287652.42309.5c
Vol. 105, No. 6, December 20071830
allodynia (6). Despite evidence of the cAMP cascade in
various pain models, the results were not consistent,
most notably in the time course of CREB activation.
The present study assessed the involvement of spinal
cAMP in nociceptive processing in a chronic neuro-
pathic pain model.
METHODS
This study was approved by the Committee of Insti-
tutional Animal Care and Use. Experiments were per-
formed on adult male Sprague-Dawley rats (weighting
250–300 g) housed in pairs before surgery. Food and
water were unrestricted. After sciatic nerve ligation, the
animals were housed individually in clean bedding of
organic cellulose fiber. The wound was checked daily
and animals were excluded if there was any sign of
wound infection or dehiscence. At the end of the proto-
col, all animals were killed with an overdose of pento-
barbital administered intraperitoneally.
Animals were anesthetized with 1.5% to 2% isoflu-
rane delivered via a nose cone (OH Medical Anesthet-
ics) and were placed in prone position. After skin
sterilization, a small incision was made in the atlanto-
occipital membrane and a polyethylene catheter (PE-5,
Becton-Dickinson, Sparks, MD) was inserted 8.5 cm.
The external portion of the catheter was secured to the
muscle at the back of the neck, and the wound was
closed with sutures. Before sciatic nerve ligation, the
function of the catheter was verified by i.t. injection of
2% xylocaine (10
L). The catheter was appropriately
placed if loss of motor power in the lower extremities
was observed. Animals with any neurological deficits
were excluded from the study. One week after i.t.
catheterization, all rats were pretested for nociceptive
baseline in response to von Frey filaments as well as
radiant heat stimulation at the proximal part of the
hindpaw (7–10).
Withdrawal responses to mechanical stimulation
were determined using a calibrated Electronic von
Frey Anesthesiometer (Model 2290CE, IITC Inc., CA)
applied from beneath the cage through openings (12
12 mm) in the plastic mesh floor to the distal portion
of the plantar aspect of the hindpaw. The stimulation
was applied starting from 0 g and continuing until a
withdrawal response was observed or a cutoff value
(70 g) was reached. This was repeated three times with
at least a 5-min test-free period between withdrawal
responses. The lowest forces from the three tests
producing a response were considered the withdrawal
threshold. Finally, the flexible von Frey filaments
above and below were tested to confirm the with-
drawal threshold. For the withdrawal latencies to heat
stimulation, rats were placed individually on an el-
evated plastic mesh floor covered with a clear plastic
cage top (21 27 15 cm) and were assessed using a
focused radiant heat source (Model-33 Tail Flick An-
algesia Meter, IITC Inc., CA). The heat stimulus, a
50-W projector lamp with an aperture diameter of 6
mm, was applied from beneath a heat-tempered glass
floor (3-mm thick) on the distal portion of the plantar
hindpaw. Paw withdrawal latencies (PWL) were mea-
sured to the nearest 0.1 s. Three trials, 5–10 min apart,
were used to obtain the average PWL. All rats were
reanesthetized with isoflurane after the average PWL
was attained. The right sciatic nerve was exposed high
on the thigh and half of the nerve was tightly ligated
with 6 0 polydioxanone suture (Ethicon, Arista, NY,
NY) as described by Seltzer et al. (7). In sham animals,
the sciatic nerve was exposed without ligation. Muscle
and skin layers were then closed. Immediately after
surgery, rats were randomly assigned into two differ-
ent groups (n 8 per group), an adenylate cyclase
inhibitor, SQ22536 (0.7
mol, dissolved and diluted
with distilled water, Biomal, Plymouth Meeting, PA)
or the same volume of distilled water (vehicle) was
administrated i.t. After a recovery time of 2 h, re-
sponses to the mechanical stimulus and radiant heat
were retested. The responses to these stimuli were
determined for the next 3, 7, and 14 days in all groups.
A pilot dose-response test was performed 24 h after
sciatic nerve ligation. Rats were randomly assigned into
different groups (n 6 8 per group) and received i.t.
SQ22536 (0.1, 0.35, 0.7, or 1
mol). The withdrawal
threshold including preligation threshold (before), 24 h
after nerve ligation (baseline) and every 30-min interval
for 3 h after drug injection (after) were determined using
a calibrated Electronic von Frey Anesthesiometer. The
percentages of maximum possible effect, where %
100 (After Baseline)/(Before Baseline) of each
dose were calculated as 20.4%, 25.8%, 55.0%, 57.6%, and
16.2% for vehicle, respectively (Fig. 1C). This result was
in accordance with that of a study in which 0.715
mol
of i.t. SQ22536 was shown to increase the mechanical
withdrawal threshold induced by IM acid injection (3).
The dose of 0.7
mol was then chosen for the present
study.
To observe the parallel changes in immunoreactiv-
ity (IR) of CREB-IR and its phosphorylated proteins
(pCREB-IR) in the dorsal horn of the spinal cord after
drug injections, different groups of animals were
killed for immunohistochemistry at 2 h, 3, 7, and 14
days after PSNL. The sham-operated animals were
used as controls. Rats were deeply anesthetized with
pentobarbital (100 mg/kg, i.p.) and perfused intracar-
dially with cold saline followed by 4% paraformalde-
hyde in 0.1 M phosphate buffer saline (PBS, pH 7.4).
The L3– 6 spinal cord segments were removed and
fixed in the above fixative for 3– 6 h. All tissues were
cryoprotected by transferring to 30% sucrose in 0.1 M
PBS at 4°C for 24 h. The spinal cord segments were cut
on a cryostat at a 20-
m thickness. The free-floating
sections were collected in PBS and blocked with 3%
normal goat serum (Vector Laboratories, Burlingame,
CA) in 0.3% Triton X-100 and 0.3% hydrogen peroxide
for1hatroom temperature. Sections were incubated
for 36 h at 4°C in a rabbit polyclonal anti-CREB
Vol. 105, No. 6, December 2007 © 2007 International Anesthesia Research Society 1831
antibody and anti-pCREB antibody (1:1000, New En-
gland Biolabs, Beverly, MA), and were processed in
biotinylated goat antirabbit IgG (1:200) using Elite
Vectastain ABC kit (Vector lab, Burlingame, CA).
Finally, the immunoprecipitates were developed with
0.05% diaminobenzedine in PBS for 3–10 min.
Eight L4 –L5 spinal cord sections were randomly
selected from each rat. Images of both ipsilateral and
contralateral dorsal horns were captured at 250
magnification using a digital camera (Nikon DXM
1200 with Eclipse E800). Anatomical landmarks were
used to identify laminar borders in gray matter of the
spinal cord in reference to the standard anatomical
mapping. The number of digitized pixels overlaid
CREB-IR and pCREB-IR cells in the superficial lami-
nae of the dorsal horn were measured automatically
Figure 1. Behavioral changes after partial sciatic
nerve ligation in rats. (A) The thermal with-
drawal latency (sec) at the hindpaw was signifi-
cantly decreased at all intervals compared with
control group (*P 0.05). Three days after
intrathecal (i.t.) injection of cyclic adenosine
monophosphate inhibitor (SQ22536), the with-
drawal threshold at the hindpaw was signifi-
cantly increased compared with the vehicle
group (#P 0.05). (B) The mechanical with-
drawal threshold (g) at the hindpaw was signifi-
cantly decreased at all intervals compared with
the control group (*P 0.05). Three days after i.t.
injection of SQ22536, the withdrawal threshold
was significantly increased compared with the
vehicle group (#P 0.05). Mean sem, n 8in
each group. (C) Dose–response curve of i.t.
SQ22536 on partial sciatic nerve ligation-induced
mechanical hyperalgesia. Peak effects were used
to calculate percentages of the maximum pos-
sible effect (%MPE). Mean sem, n 8 in each
group.
1832
CREB and Chronic Neuropathic Pain ANESTHESIA & ANALGESIA
using image analysis software (Image-Pro Plus, Media
Cybernetics, Inc., GA Avenue, Silver Spring). The
values from these sections were averaged for each
animal. To examine possible changes in the expression
of CREB-IR and pCREB-IR within cells, the mean
optical density of all positive (supra-threshold) objects
were compared.
Different groups of animals were killed for Western
immunoblots and the lumbar spinal cords (L4–5) were
excised. The dorsal halves were weighed, homoge-
nized and centrifuged at 7000g for 15 min. The 50 mM
Tris–HCl (pH 7.4) homogenizing buffer contained
with 150 mM NaCl, 50 mM NaF, 2 mM EDTA, 0.25%
sodium deoxycholate, 1% Nonidet P-40, and 5
g/mL
of a mixture of protease inhibitors included bestatin,
4-(2-aminoethyl) benzene-sulfonyl fluoride, pepstatin
A, aprotinin, and leupeptin. Total protein content in
the homogenates was determined and separated by
SDS-PAGE electrophoresis (15
g of total protein per
lane), transferred onto nitrocellulose membranes, and
blocked with PBS solution containing nonfat, dry milk
(5%) for 1 h. The membranes were first incubated with
rabbit polyclonal antibodies to CREB (1:1000) and
pCREB (1:500) overnight at 4C, and then in secondary
antibody at 1:10000 (Jackson, PA) for 1 h at room
temperature. The signals were visualized by chemilu-
minescence and quantified using PharosFX™ Plus
Molecular Imager® System (BIO-RAD Laboratories
Inc. Hercules, CA). Values are expressed as mean
sem. Groups were compared using one-way analysis
of variance with Student Newman–Keuls multiple
comparisons. P 0.05 was considered significant.
RESULTS
After PSNL, most rats developed tactile allodynia and
thermal hyperalgesia in the ipsilateral hindpaw, com-
pared with the control group for at least 14 days. The
allodynia and hyperalgesia in the ipsilateral hindpaw
was significantly attenuated 1–3 days after i.t. injection of
SQ22536, when compared with vehicle (Fig. 1). Increases
in CREB-IR and pCREB-IR cell profiles were also found
in the dorsal horn of L4–5 spinal cord with predomi-
nance in the superficial layers (Fig. 2). The mean pixel
number, optical density, and protein expression in both
groups were significantly higher than that of the prein-
jured baseline. However, differences in the total
CREB-IR cells were not observed between SQ22536 and
Figure 2. Photomicrographs of total cyclic adenosine monophosphate response element-binding protein (CREB) and
phosphorylated CREB-immunoreactivity (CREB-IR and pCREB-IR) cells in the dorsal horn of L4–5 spinal cord of rats after
intrathecal (i.t.) injection of vehicle (A1–A5, C1–5) or SQ22536 (B1–B5, D1–D5). After partial sciatic nerve ligation, abundant
total CREB-IR and pCREB-IR cells were observed in the dorsal horn compared with baseline (A1, B1, C1, and D1). No
significant difference in total CREB-IR cells was observed between the vehicle (A2–A5) and SQ22536 (B2–B5) injected rats.
However, after i.t. injection of SQ22536, the number of total pCREB-IR cells (D2–D5) was markedly reduced in the dorsal horn
compared with vehicle-treated rats (C2–5). The reduction of total pCREB-IR cells in the dorsal horn of SQ22536-treated rats
was more evident in the first 3 days. Scale BAR 50
m.
Vol. 105, No. 6, December 2007 © 2007 International Anesthesia Research Society 1833
vehicle control (Figs. 3 and 4A). After i.t. SQ22536,
pCREB-IR cell profiles were partially reduced, but not by
vehicle, in the ipsilateral dorsal horn. Quantitatively, the
mean pixel number, optical density and protein expres-
sion after i.t. SQ22536 were significantly decreased in
comparison with control, especially after the first 3 days
(Figs. 4B and 5).
DISCUSSION
The results of this study demonstrated that PSNL
significantly induced an increase in the phosphoryla-
tion of CREB for 2 wk. Furthermore, the expression of
pCREB in cells of the superficial dorsal horn was
correlated to the reduction in the mechanical and
thermal withdrawal thresholds, indicating that behav-
ioral changes were associated with an increase in
pCREB. This result also showed that the increased
pCREB depended on the activation of the cAMP
pathway, as it could be prevented for at least 3 days by
blocking the adenylate cyclase activity.
According to previous reports, activation of the
cAMP pathway in the spinal cord was implicated in the
mediation of nociceptive processing. Mechanical hyper-
algesia was produced by spinal activation of the cAMP
pathway (4,5), and spinal activation of adenylate cyclase
increased the activities of neurons in the spinothalamic
tract in response to pinch stimulation, which was
blunted by pretreatment with a PKA inhibitor (11). Mice
lacking adenylate cyclases manifested a reduction in
behavioral responses to formalin or complete Freund’s
adjuvant stimuli (12). Additionally, blocking adenylate
cyclase or PKA prevented the mechanical hyperalgesia
and allodynia induced by capsaicin injection (4,5). In the
present study, PSNL-induced tactile allodynia and ther-
mal hyperalgesia were attenuated by blockade of the
cAMP pathway in the early phase.
Our results showed that PSNL significantly induced
the expression of CREB, a transcription factor, at least for
2 wk, whereas i.t. adenylate cyclase inhibitor had no
significant effect. Increases in CREB resulted in a greater
Figure 3. The quantification of total cyclic aden-
osine monophosphate response element-binding
protein (CREB)-immunoreactivity (CREB-IR) cells
in the dorsal horn of SQ22536 or vehicle-treated
rats. (A) After partial sciatic nerve ligation, the
mean pixel number of total CREB-IR cells in-
creased in the dorsal horn compared with the
prelesion value (*P 0.05). No significant
difference in the total CREB-IR cell count
was observed between the SQ22536 and
their counterparts in vehicle-treated rats. (B)
The mean optical density in the dorsal horn
of SQ22536 or vehicle-treated rats was also
significantly increased compared with the
prelesion value (*P 0.05). No significant
difference in the total CREB-IR optical den-
sity was observed between the SQ22536 and
their counterparts in vehicle-treated rats.
Mean sem, n 8 in each group.
1834
CREB and Chronic Neuropathic Pain ANESTHESIA & ANALGESIA
resource of protein for phosphorylation. Previous stud-
ies reported that CREB mRNA and CREB-IR caused an
increase in hippocampus after chronic administration of
antidepressants in rats (13). Little information is avail-
able on the effect of chronic noxious stimulation on the
expression of CREB, which is still controversial. The
results of Sluka’s study revealed an increase in CREB
level 24 h, but not in 1 wk, after repeated IM injections of
acid in rats (3). In contrast, Miletic et al. (14) reported that
there were no differences in the content of CREB be-
tween the PSNL model and control. Much work has still
to be accomplished in the elucidation of different mecha-
nisms underlying the control of the CREB expression in
chronic pain disorders.
When CREB is phosphorylated, it binds to specific
DNA consensus sequences such as cAMP responsive
element, and regulates immediate-early genes, includ-
ing c-fos (15) and c-jun (16). Some late effector genes
such as those that transcribed dynorphin (17), sub-
stance P receptor NK 1 (18), and other elements (19)
were also involved. Different animal models showed
an association between the induction of nociception
and the expression of pCREB at various intervals. For
instance, an increase in the expression of pCREB
occurred at the hindpaw of rats after subcutaneous
injection of carrageenan (12), formalin or complete
Freund’s adjuvant (12,18,20). Animals with repeated
IM injection of acid (3) and those that suffered from
neuropathic pain demonstrated a similar phenom-
enon (2,14). The expression of phosphorylated CREB
is involved in the temporal effects of hyperalgesia in
neuropathic (14) and inflammatory pain (20). Further-
more, the amount of phosphorylated CREB seemed to
be stimulus dependent. Increasing the volume of
formalin injected into the hindpaw resulted in an
increase in phosphorylated CREB (20). Phosphoryla-
tion of CREB was also increased in rats with morphine
tolerance as well as in CREB mutant mice, whose main
symptoms of morphine withdrawal were significantly
attenuated (21). Taken together, these observations
strongly suggested that increased phosphorylation of
CREB was likely to be involved in the development of
inflammatory pain and neuropathic pain.
The results of previous observations also demon-
strated that different response-time courses in the
phosphorylation of CREB were demonstrated in vari-
ous pain models. For instance, Hoeger-Bement and
Sluka (3) reported that the mechanical hyperalgesia
and phosphorylation of CREB depended on the early
activation of the cAMP pathway during the first 24 h,
but are independent of the cAMP pathway 1 wk after
IM injection of acid. The temporal changes in the
phosphorylation of CREB was observed in the follow-
ing chronic neuropathic pain models, i.e., 2 h after
loose ligation of the sciatic nerve (22), 3 wk after PSNL
(2), and 7 days, but not 28 days after a chronic nerve
constriction model (14). The results of the present
study gave credence to the hypothesis that activation
of the cAMP pathway was likely to be involved in the
early phase of maintenance, but not the later phase of
induction, in rats with chronic neuropathic pain. In
fact, the temporal effects of cAMP pathway activation
were manifested in other models of neuroplasticity.
For instance, the early phase of long-term enhance-
ment depended on the activation of the cAMP path-
way, but was decreased by the inhibition of this
pathway (23,24). In the hippocampus, the activity of
PKA was rapidly increased in the initial stage of
spatial learning and started to decrease when the
Figure 4. The expression of cyclic adenosine monophosphate response element-binding protein (CREB) (A) and pCREB (B)
proteins in the spinal cord of rats in groups: control, sham, sham-operated with vehicle (Sham Veh), partial sciatic nerve
ligation with vehicle (PSNL Veh), PSNL with SQ22536 (PSNL SQ22536). For equal protein loading, membranes were
reprobed for b-actin using mouse monoclonal antibody. The intensity of the bands was analyzed using densitometry and
plotted as histograms, as shown in each panel. Data shown are from three independent experiments (n 6); error bars
indicate sem.*P 0.05 compared with control and Sham; #P 0.05 compared with PSNL Veh.
Vol. 105, No. 6, December 2007 © 2007 International Anesthesia Research Society 1835
activity of protein kinase C had reached to the maxi-
mum at the later stage (25). Previous evidence indi-
cates that protein kinase C activation seemed to be
critical in the maintenance phase of long-term en-
hancement such as memory (26). Although the
results of the present study were compatible with
the above phenomenon, further verification is still
needed.
Consistent with our results, a previous study dem-
onstrated a reduction in hyperalgesia secondary to the
inhibition of the cAMP pathway at 24 h, but not 1 wk,
in a repeated IM acid injection pain model (3).
Changes in pCREB at 24 h were also found to have
strong correlation with an increase in the mechanical
withdrawal threshold, suggesting a role for CREB
phosphorylation in the early phase of maintenance in
mechanical hyperalgesia induced by IM acid injection.
As demonstrated in our study, CREB phosphorylation
in the early phase of neuropathic pain was mediated
by the activation of the cAMP pathway because of the
reversal of PSNL-induced pCREB enhancement sec-
ondary to cAMP inhibition. Although the role of
phosphorylated CREB in the maintenance of neuro-
pathic pain has been confirmed in the present study,
other confounding factors underlying its mechanisms
still need to be considered. For instance, in addition to
PKA, a number of intracellular messengers resulting
to the phosphorylation of CREB, such as calmodulin-
dependent protein kinase, nerve growth factor-
mediated Ras/Raf mitogen-activated protein kinase
kinase-1/2 and extracellular regulated kinase,
mitogen-activated protein kinase, p38, and other ki-
nases may contribute to the regulation of CREB phos-
phorylation (1,27,28). One limitation of our study was
the role of the cAMP-CREB pathway because the
inhibition of cAMP only attenuated the PSNL-induced
allodynia or hyperalgesia, but not the paw withdrawal
threshold in response to mechanical and heat stimu-
lation. This observation suggested that pathways
other than cAMP-CREB may be involved in the
Figure 5. The quantification of total phos-
phorylated cyclic adenosine monophosphate
response element-binding protein (CREB)-
immunoreactivity (pCREB-IR) cells in the
dorsal horn of rats treated with intrathecal
(i.t.) SQ22536 or vehicle control. (A) After
partial sciatic nerve ligation, the mean pixel
number of total pCREB-IR cells increased in
the dorsal horn compared with the prelesion
value (*P 0.05). After i.t. injection of
SQ22536, the mean pixel number of total
pCREB-IR cells was reduced in the dorsal
horn compared with their counterparts in
vehicle-treated rats. The reduction of total
pCREB-IR cells in the dorsal horn of
SQ22536-treated rats was significantly dif-
ferent in the first 3 days (#P 0.05). (B)
Mean optical density in the dorsal horn of
SQ22536 or vehicle-treated rats was also
significantly increased compared with the
prelesion value (*P 0.05). After i.t. injec-
tion of SQ22536, the mean optical density of
total pCREB-IR cells was reduced compared
with their counterparts in vehicle-treated
rats. The reduction of total pCREB-IR optical
density in the dorsal horn of SQ22536-
treated rats was significantly different in the
first 3 days (#P 0.05). Mean sem, n 8
in each group.
1836
CREB and Chronic Neuropathic Pain ANESTHESIA & ANALGESIA
mechanisms underlying the early phase of PSNL-
induced tactile allodynia and thermal hyperalgesia.
Our results demonstrated that CREB and the phos-
phorylation of CREB in the spinal cord significantly
increased for 2 wk after PSNL in parallel with an
increase in the mechanical and thermal withdrawal
threshold. The increase in pCREB level was partially
reversed by the blockade of the cAMP pathway early
in the 3 days. Our results strongly imply that increases
in pCREB contributed to PSNL-induced tactile allo-
dynia and thermal hyperalgesia. Modulation of cAMP
pathway may be of clinical importance if early inter-
ventions can be performed in patients with chronic
neuropathic pain.
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    • "This is in consistent with our previous finding that nerve injury induces hypofunction of glutamatergic activity in the PAG (Ho et al. 2013). This antinociceptive role of AC in the PAG is in contrast to the pronociceptive role of AC in the ACC or spinal dorsal horn (Wei et al. 2002; Liou et al. 2007). The discrepancy is unknown but different subtypes of AC may regulate pain conversely. "
    [Show abstract] [Hide abstract] ABSTRACT: Neuropathic pain has been attributed to nerve injury-induced elevation of peripheral neuronal discharges and spinal excitatory synaptic plasticity while little is known about the contribution of neuroplasticity changes in the brain stem. Here, we examined synaptic plasticity changes in the ventrolateral periaqueductal gray (vlPAG), a crucial midbrain region for initiating descending pain inhibition, in spinal nerve ligation (SNL)-induced neuropathic rats. In vlPAG slices of sham-operated rats, forskolin, an adenylyl cyclase (AC) activator, produced long-lasting enhancement of excitatory postsynaptic currents (EPSCs). This is a presynaptic effect since forskolin decreased the paired-pulse ratio and failure rate of EPSCs, and increased the frequency, but not amplitude of miniature EPSCs. Forskolin-induced EPSC potentiation was mimicked by a β-adrenergic agonist (isoproterenol), and prevented by an AC inhibitor (SQ22536) and a cAMP-dependent protein kinase (PKA) inhibitor (H89), but not by a phosphodiesterase (PDE) inhibitor (Ro 20-1724) or an A1-adenosine antagonist (DPCPX). Both forskolin- and isoproterenol-induced EPSC potentiation was impaired in PAG slices of SNL rats. The SNL group had lower AC, but not PDE, activity in PAG synaptosomes than the sham group. Conversely, IPSCs in vlPAG slices were not different between SNL and sham groups. Intra-vlPAG microinjection of forskolin alleviated SNL-induced mechanical allodynia in rats. These results suggest that SNL leads to inadequate descending pain inhibition owing to impaired glutamatergic synaptic plasticity mediated by the AC-cAMP-PKA signaling, possibly due to AC down regulation, in the PAG, leading to long-term neuropathic pain. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Apr 2015
    • "cAMP signalling has been primarily implicated in the development of pain hypersensitivity in an inflammatory state as many inflammatory mediators signal through G protein-coupled receptors that are coupled with G proteins that activate (Gs) adenylate cyclase, whilst other inflammatory mediators may cause an increase in intracellular calcium that activates calcium-sensitive adenylate cyclases. Pharmacological and genetic evidences also suggest a role for cAMP in the development of mechanical allodynia in rodent models of neuropathic pain [18, 22, 23]. For example, the development of mechanical allodynia is severely impaired in models of neuropathic pain in mice deficient for adenylate cyclase 5 [18]. "
    [Show abstract] [Hide abstract] ABSTRACT: Mechanical allodynia (other pain) is a painful sensation caused by innocuous stimuli like light touch. Unlike inflammatory hyperalgesia that has a protective role, allodynia has no obvious biological utility. Allodynia is associated with nerve damage in conditions such as diabetes, and is likely to become an increasing clinical problem. Unfortunately, the mechanistic basis of this enhanced sensitivity is incompletely understood. In this review, we describe evidence for the involvement of candidate mechanosensitive channels such as Piezo2 and their role in allodynia, as well as the peripheral and central nervous system mechanisms that have also been implicated in this form of pain. Specific treatments that block allodynia could be very useful if the cell and molecular basis of the condition could be determined. There are many potential mechanisms underlying this condition ranging from alterations in mechanotransduction and sensory neuron excitability to the actions of inflammatory mediators and wiring changes in the CNS. As with other pain conditions, it is likely that the range of redundant mechanisms that cause allodynia will make therapeutic intervention problematic.
    Full-text · Article · May 2014
    • "The down-stream effectors of the adenylate cyclase/cAMP pathway were evaluated by determining the phosphorylation of the transcription factor CREB (pCREB) at Ser133 (Gonzalez et al., 1989), which is upregulated in numerous animal models for pain (Hoeger-Bement and Sluka, 2003; Ji and Rupp, 1997; Liou et al., 2007). Consistent with the decrease in cAMP levels, light exposure Fig. 2. Photostimulation of optoMOR inhibits Ca 2 þ influx. "
    [Show abstract] [Hide abstract] ABSTRACT: The use of opioids, which achieve therapeutic analgesia through activation of μ-opioid receptors, are limited in the management of chronic pain by adverse effects including tolerance and addiction. Optogenetics is an emerging approach of designing molecular targets that can produce cell-specific receptor-mediated analgesia with minimal side effects. Here we report the design and functional characterization of a chimeric μ-opioid receptor that could be photoactivated to trigger intracellular signaling. A prototype optoactive μ-opioid receptor (optoMOR) was designed by replacing the intracellular domains from rhodopsin with those of the native μ-opioid receptor and was transiently expressed in human embryonic kidney (HEK293) cells. Expression and distribution of the protein were confirmed by immunocytochemistry. The signal-transduction mechanisms induced by photoactivation of the optoMOR were evaluated and compared with the native μ-opioid receptor stimulation by an agonist, D-Ala2, N-MePhe4, Gly-ol-enkephalin (DAMGO). Cells were depolarized by extracellular potassium and the depolarization-induced calcium (Ca2+) influx was quantified by using Fura-2 imaging. The forskolin-stimulated adenylate cyclase/cAMP cascade was evaluated by ELISA or western blotting of brain-derived neurotrophic factor (BDNF) and the phosphorylation of cAMP response element binding protein (CREB). The optoMOR protein distribution was observed intracellularly and on the plasma membrane similar to the native μ-opioid receptor in HEK293 cells. Photoactivation of optoMOR decreased the Ca2+ influx and inhibited the forskolin-induced cAMP generation, activation of CREB, and BDNF levels in optoMOR-expressing cells similar to the activation of native μ-opioid receptor by DAMGO. Thus the current study has accomplished the design of a prototype optoMOR and characterized the cellular signaling mechanisms activated by light stimulation of this receptor.
    Full-text · Article · Feb 2013
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