Molecular Mechanisms Underlying the Enhanced
Analgesic Effect of Oxycodone Compared to Morphine in
Chemotherapy-Induced Neuropathic Pain
Karine Thibault1,2,3*¤, Bernard Calvino1,2, Isabelle Rivals4, Fabien Marchand5,6, Sophie Dubacq1,2,
Stephen B. McMahon3, Sophie Pezet1,2
1 Brain Plasticity Unit, ESPCI-ParisTech, Paris, France, 2 Centre National de la Recherche Scientifique, UMR 8249, Paris, France, 3 Neurorestoration Group, The Wolfson
Centre for Age-Related Diseases, King’s College London, London, United Kingdom, 4 Equipe de Statistique Appliquée, ESPCI-ParisTech, Paris, France, 5 Institut National de
la Santé et de la Recherche Médicale, Unité 1107, NEURO-DOL, Clermont-Ferrand, France, 6 Clermont Université, Université d’Auvergne, Pharmacologie Fondamentale et
Clinique de la Douleur, Clermont-Ferrand, France
Abstract
Oxycodone is a m-opioid receptor agonist, used for the treatment of a large variety of painful disorders. Several studies have
reported that oxycodone is a more potent pain reliever than morphine, and that it improves the quality of life of patients.
However, the neurobiological mechanisms underlying the therapeutic action of these two opioids are only partially
understood. The aim of this study was to define the molecular changes underlying the long-lasting analgesic effects of
oxycodone and morphine in an animal model of peripheral neuropathy induced by a chemotherapic agent, vincristine.
Using a behavioural approach, we show that oxycodone maintains an optimal analgesic effect after chronic treatment,
whereas the effect of morphine dies down. In addition, using DNA microarray technology on dorsal root ganglia, we provide
evidence that the long-term analgesic effect of oxycodone is due to an up-regulation in GABAB receptor expression in
sensory neurons. These receptors are transported to their central terminals within the dorsal horn, and subsequently
reinforce a presynaptic inhibition, since only the long-lasting (and not acute) anti-hyperalgesic effect of oxycodone was
abolished by intrathecal administration of a GABAB receptor antagonist; in contrast, the morphine effect was unaffected.
Our study demonstrates that the GABAB receptor is functionally required for the alleviating effect of oxycodone in
neuropathic pain condition, thus providing new insight into the molecular mechanisms underlying the sustained analgesic
action of oxycodone.
Citation: Thibault K, Calvino B, Rivals I, Marchand F, Dubacq S, et al. (2014) Molecular Mechanisms Underlying the Enhanced Analgesic Effect of Oxycodone
Compared to Morphine in Chemotherapy-Induced Neuropathic Pain. PLoS ONE 9(3): e91297. doi:10.1371/journal.pone.0091297
Editor: Michael Costigan, Boston Children’s Hospital and Harvard Medical School, United States of America
Received October 1, 2013; Accepted February 7, 2014; Published March 11, 2014
Copyright: ß 2014 Thibault et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the ‘‘Institut UPSA de la douleur’’ and the ‘‘Centre National de la Recherche Scientifique’’, CNRS. KT received a two year
doctoral fellowship from Mundipharma laboratory (ML). KT received one year of post-doctoral fellowship from ‘‘Fondation pour la Recherche Medicale’’. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: karine.thibault@parisdescartes.fr
¤ Current address: Glia-Glia & Glia-Neuron Interactions in Neurophysiopathology Group, CNRS FR3636, Paris Descartes University, Paris, France
of pain, including visceral pain [5], post-operative pain [6] and
cancer pain [7,8]. Moreover, the oxycodone-paracetamol combination was shown to be effective against chronic lower back pain
[9], whereas morphine treatment did not show any beneficial
effect [10]. In patients with neuropathic pain, oxycodone
treatment (mostly in combination with anticonvulsants) improves
health-related quality of life and diminishes the impact of pain on
physical activity and sleep [11]. However, the neurobiological
mechanisms underlying the therapeutic action of these two opioids
are currently only partially understood. The aim of this study was
to identify the molecular changes that produce the different
analgesic effects of morphine and oxycodone.
Vincristine is an anti-neoplasic drug used to treat a wide variety
of cancers. Nonetheless, it induces a peripheral neurotoxicity and
subsequent neuropathic pain in both patients [12,13] and animal
models [14–17]. Here, we observed in an animal model of
vincristine-induced neuropathic pain that oxycodone has a longer
lasting analgesic effect than morphine. Based on DNA microarray
Introduction
Approximately 7–8% of the European population suffers from
neuropathic pain, and 5% of these cases may be severe [1].
Unfortunately, the pharmacotherapy of neuropathic pain is
frequently unsatisfactory for patients. One reason for their poor
efficacy is the lack of a clear understanding of the neurobiological
mechanisms that underlie neuropathic pain. Morphine is the most
commonly used opioid analgesic in the past 30 years. However,
repeated use of opioids induces a tolerance phenomenon, which
significantly reduces their analgesic effects. Among clinically used
opioids, oxycodone shows excellent anti-hyperalgesic and antiallodynic effects against neuropathic pain [2,3]. Oxycodone is a
semi-synthetic opioid analgesic, and has been used in clinics since
1917 [4]. Like other opioids, including morphine, the analgesic
effect of oxycodone is mainly mediated through the activation of
the m-opioid receptor. Interestingly, the analgesic effect of
oxycodone seems to be as potent as morphine on different types
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Oxycodone and Pain Relief
the injection of saclofen (10 ml; Ref S166, Sigma-Aldrich, St.
Louis, MO) or saline. Saclofen was diluted to a concentration of
1 mg/ml in saline. This dose was chosen according to [21,22].
Twenty-four rats received i.p. injections of vincristine. At D15, rats
were subdivided into two groups: 12 rats received chronic
injections of oxycodone, and the other 12 received chronic
injections of saline (over a 5 day-treatment). At D19, each group
was further divided into 2 sub-groups that received an i.t. injection
of saclofen or saline (n = 6 vincristine-oxycodone-saclofen treated
rats; n = 6 vincristine-oxycodone-saline treated rats; n = 6 vincristine-saline-saclofen treated rats; n = 6 vincristine-saline-saline
treated rats).
technology in dorsal root ganglia, we report an important list of
genes that are differentially expressed in oxycodone-treated rats, as
compared to morphine-treated subjects. This provides clues on the
mechanisms underlying their different long-term actions. Furthermore, we provide direct evidence for an involvement of GABAB
receptors in the long-lasting effect of oxycodone on neuropathic
pain.
Materials and Methods
All experiments were performed in agreement with the
European Community Council Directive of 24 November 1986
(86/609/EEC) and with guidelines on ethical standards for the
investigation of experimental pain in animals, published in a Guest
Editorial in the journal ‘Pain’ (Committee for Research and
Ethical issues of the I.A.S.P., 1980). This study was approved by
the ethics committee C2EA-59: ‘Comité d’éthique en matière
d’expérimentation animale Paris Centre et Sud’. Accordingly to
the rule of the ‘3R’ of the European regulation for the use of
laboratory animals, the number of animals was kept to the
minimum possible. Experiments were performed on 123 male
Sprague Dawley rats (Janvier, Le Genest St Isle, France) weighing
150–175 g at the beginning of the experiments. Great care was
taken, particularly with regard to housing conditions, to avoid or
minimize discomfort of the animals: rats were housed four in a
cage to minimize the possibility of painful interactions. The
animals were kept on solid floor cages with a deep layer of sawdust
to accommodate the excess of urination and cages were changed
three times a week. They were kept at a constant temperature of
22uC, with a 12 h alternative light/dark cycle. Food and water
were available ad libitum.
Influence of the GABAB receptor on the analgesic effect
of acute injection of morphine and oxycodone and
repeated injections of morphine: intrathecal
administration of saclofen
Thirty-six rats received i.p. vincristine injections. The role of the
GABAB receptor on the analgesic effect of acute injection of
opioids was tested at D15 (n = 6 vincristine-oxycodone-saclofen
treated rats; n = 6 vincristine-oxycodone-saline treated rats; n = 6
vincristine-morphine-saclofen treated rats; n = 6 vincristine-morphine-saline treated rats; n = 6 vincristine-saline-saclofen treated
rats; n = 6 vincristine-saline-saline treated rats). The role of the
GABAB receptor on the analgesic effect of chronic injections of
morphine was tested at D19, as with oxycodone (see above).
Behavioural tests
The animals received a two-week habituation period prior to
the start of the peripheral neuropathy-inducing vincristine
injections, as previously described [23].
Chronic effects of analgesic treatment. Behavioural tests
were performed on D1 and D15 of vincristine treatment in 21
vincristine-treated rats and 18 control rats. The time-course study
on the effects of oxycodone and morphine was performed at D15
and D19, in which behavioural tests were repeated 30, 60 and 90
minutes following morphine (n = 7 vincristine-morphine treated
rats; n = 6 saline-morphine treated rats), oxycodone (n = 7
vincristine-oxycodone treated rats; n = 6 saline-oxycodone treated
rats), or saline injections (n = 7 vincristine-saline treated rats; n = 6
saline-saline treated rats).
Induction of peripheral neuropathy by vincristine and
analgesic treatment
Animals were randomly chosen and tested by an experimenter
blinded to the animal’s treatment. Fifty-seven rats received
intraperitoneal (i.p.) injections of vincristine (0.1 mg/kg/day;
Oncovin-1 mg, EG LABO, Boulogne-Billancourt, France) for
two 5-day cycles with a 2-day pause between cycles, as previously
described [17]. Thirty control rats received i.p. injections of the
vehicle (saline: 9% NaCl) according to the same protocol. At the
end of vincristine treatment (D15), each group was divided into 3
sub-groups that received chronic injections of morphine (3.33 mg/
kg diluted in saline, i.p. injection; laboratoire Cooper, France),
oxycodone (3.33 mg/kg diluted in saline, i.p. injection; OxyNorm,
Napp Pharmaceuticals Ltd, Cambridge, UK) or saline during 5
days (D19). These doses were chosen according to a previous
report [18], as well as unpublished preliminary data showing an
equipotent analgesic effect of morphine and oxycodone on
mechanical hyperalgesia (pinch test) in naive animals, when used
at 3.33 mg/kg.
GABAB receptor antagonist study on the long-lasting
analgesic
effect
of
repeated
injections
of
oxycodone. Behavioural tests were performed on D1 and D15
of vincristine treatment in 24 vincristine-treated rats. Behavioural
tests were performed on D15 as a control for the development of
neuropathic pain state; the animals were then repeatedly treated
with oxycodone. The rats received one injection of oxycodone or
saline at D19, and were then tested 30 minutes later as a control
for the long-lasting analgesic effect of oxycodone (D19). Four
hours after this first injection, rats were injected again with
oxycodone or saline (D199). Fifteen minutes after this second
injection, rats were anesthetized with isoflurane (Baxter, Maurepas, France) and injected intrathecally with saclofen (10 mg; Ref
S166, Sigma-Aldrich, St. Louis, MO) diluted in saline. Rats were
behaviourally tested once again, 15 minutes after the saclofen
injection (n = 6 vincristine-oxycodone-saclofen treated rats; n = 6
vincristine-oxycodone-saline treated rats; n = 6 vincristine-salinesaclofen treated rats; n = 6 vincristine-saline-saline treated rats).
Influence of the GABAB receptor on the long-lasting
analgesic effect of repeated oxycodone injections:
intrathecal administration of saclofen
This experiment was performed to evaluate the involvement of
GABAB receptors in the analgesic effect of oxycodone. Intrathecal
injections were performed by lumbar puncture, as previously
described [19,20]. Briefly, a rat was slightly anesthetized with
isoflurane gas (Baxter, Maurepas, France) and held in one hand by
the pelvic girdle; a 25-gauge x1-inch needle connected to a 10-ml
Hamilton syringe was then inserted into the subarachnoidal space
between the spinous processes of L5 and L6, until a tail-flick was
elicited. The syringe was held in position for several seconds after
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GABAB receptor antagonist study on the analgesic effect
of acute injection of oxycodone or morphine and chronic
injections of morphine. Behavioural tests were performed on
D1 of vincristine treatment in 36 vincristine-treated rats. The rats
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Oxycodone and Pain Relief
Thibault et al. [24]. First, a stimulus was applied with a smooth
(marten hair) paint-brush to each hind paw, five times within a
five-second interval. Each stimulus that resulted in a response was
scored as ‘1’, and the absence of a response was scored as ‘0’. The
10 stimulation scores were added, giving a total score between 0
and 10 for each rat. Next, the stimulation was applied with a
rough (pig bristle) paint-brush, using the same protocol. Dynamic
mechanical allodynia or hyperesthesia were defined as a significant
increase in the total score using the smooth or the rough brush,
respectively.
Statistical analysis of the behavioural data. Behavioural
data from electronic Von Frey and Pinch tests were examined
using two-way analysis of variance (ANOVA) followed by a
Student Newman–Keuls test for two-by-two comparisons. Behavioural data from paint-brush tests were examined with a Kruskal–
Wallis test followed by a Wilcoxon matches pairs test and a Mann–
Whitney U test for two-by-two comparisons. Data were expressed
as means 6 SEM, and the levels of significance were set at:
*p,0.05; **p,0.01; ***p,0.001.
received one injection of oxycodone, morphine or saline at D15.
Fifteen minutes later, rats were anesthetized with isoflurane and
injected intrathecally with saclofen (10 mg; Ref S166, SigmaAldrich, St. Louis, MO) diluted in saline. Rats were tested again 15
minutes after the saclofen injection (n = 6 vincristine-oxycodonesaclofen treated rats; n = 6 vincristine-oxycodone-saline treated
rats; n = 6 vincristine-morphine-saclofen treated rats; n = 6
vincristine-morphine-saline treated rats; n = 6 vincristine-salinesaclofen treated rats; n = 6 vincristine-saline-saline treated rats). At
D19 (i.e. 5 days after the start of opioids treatment), the same
experiment was performed following chronic injections of morphine (n = 6 vincristine-morphine-saclofen treated rats; n = 6
vincristine-morphine-saline treated rats; n = 6 vincristine-salinesaclofen treated rats; n = 6 vincristine-saline-saline treated rats).
For ease of reading, we have presented the results in two separate
figures: the effect of saclofen after acute injection of oxycodone,
and the effect of saclofen after morphine treatment.
Assessment of static mechanical sensitivity. The static
mechanical allodynia was measured using the electronic Von Frey
test, as previously described [23]. Briefly, an Electronic Von Frey
hair unit (EVF-3, Bioseb, Chaville, France) was used to measure
the sensitivity threshold in one test; measurements ranged from 0
to 500 grams with a 0.2 gram accuracy. Paw sensitivity threshold
was defined as the minimal pressure required to elicit a robust and
immediate withdrawal reflex of the paw. The stimulus was applied
to each hind paw five times within a five-second interval; by
convention, the threshold for each rat was the average of ten
measured values. Static mechanical allodynia was defined as a
significant decrease in withdrawal thresholds to Von Frey
application.
The results for each group in the GABAB receptor antagonist
study were expressed as follows: d-paw withdrawal threshold (%6
S.E.M.),
calculated
from individual
thresholds:
paw0 withdrawal
ðT30{T0Þ
ðT30 {T0Þ
|100 at D19 and
|100 at D199.
T0
T0
The effect of saclofen on mechanical allodynia was defined as a
significant decrease in d-paw withdrawal threshold to Von Frey
application.
Assessment of static mechanical hyperalgesia. Static
mechanical hyperalgesia was measured using the ‘Pinch’ test, as
previously described [23]. Rats were individually taken by the
experimenter and held without restraint on the experimenter’s lap
for 5 min. The threshold of static mechanical sensitivity was
determined using a ‘Pincher’ unit (Bioseb, Chaville, France).
Briefly, pressure was applied with a calibrated forceps (ranging
from 0–800 g) to the mid-plantar area of each hind paw, to elicit a
robust and immediate withdrawal reflex of the stimulated paw.
The stimulus was applied to each hind paw three times within a 2
minute-interval; by convention, the threshold for a rat was the
average of the six measured values. Static mechanical hyperalgesia
was defined as a significant decrease in withdrawal thresholds to
pincher application.
The results for each group in the GABAB receptor antagonist
study were expressed as follows: d-paw withdrawal threshold (%6
S.E.M.),
calculated
from individual
thresholds:
paw0 withdrawal
ðT30{T0Þ
ðT30 {T0Þ
|100 at D19 and
|100 at D199.
T0
T0
The effect of saclofen on mechanical allodynia was defined as a
significant decrease in the d-paw withdrawal threshold to pincher
application.
DNA microarray analysis
RNA preparation for microarray analysis and microarray
hybridisation. Two hours after the end of the behavioural
study (D19), rats were deeply anaesthetized with pentobarbital
(60 mg/kg i.p; Ceva Santé Animale, Libourne, France) and
decapitated (n = 4 vincristine-morphine, n = 4 vincristine-oxycodone, n = 4 vincristine-saline treated rats; and n = 4 salinemorphine, n = 4 saline-oxycodone, n = 4 saline-saline rats). Lumbar dorsal root ganglia (DRGs) L2 through L5 were dissected and
immediately frozen on dry ice. Tissues were kept at 280uC until
homogenisation. Total RNA from individually dissected frozen
DRGs was extracted and treated with DNAse using the RNeasyH
Lipid Tissue Mini kit (Qiagen, Courtaboeuf, France), according to
the manufacturer’s protocol. The quality and quantity of each
RNA sample was checked using an Agilent 2100 Bioanalyzer with
RNA 6000 NanoChips (Agilent Technologies, Massy, France).
Five hundred ng of RNA were sent to the Integragen Illumina
microarray facility (Integragen, Evry, France, www.integragen.
com) for sample processing. Illumina pangenomic rat microarrays
(RatRef-12 Expression BeadChips), including 22,517 probes, were
also performed at the Integragen Illumina microarray facility
(Evry, France), according to the Illumina procedures.
DNA microarray statistical analysis. All manipulations
and statistical analyses were performed with the R freeware. For
Illumina beadchip data, all spots with a detection p-value greater
than 0.05% were excluded. A two-way ANOVA was performed,
using vincristine treatment as the first factor and analgesic
treatment as the second factor. Student t-tests were performed to
find differentially expressed genes between morphine and oxycodone treatment, in vincristine-treated rats or controls. The
reported p-values correspond to the control of the type I error
risk of each individual gene (no adjustment for multiple testing).
The data have been deposited in NCBIs Gene Expression
Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and assigned Series accession number GSE53897.
Real-time quantitative PCR. Quantitative real-time PCR
was performed using the fluorescent binding-dye SYBR Green
(Absolute Blue SYBR Green Rox mix, ABgene, Thermo
Scientific) on a real-time PCR machine (LightCycler Technology,
Roche) with primer sets designed at the Roche website (www.
roche-applied-science.com). Each qPCR assay was conducted in
triplicate, using cDNA derived from 50 ng total RNA from each
animal. Each sample well contained 4 ml of cDNA (5 ng/ml), with
a primer concentration of 0.5 mM plus 5 ml of SYBR Green. Each
Assessment of dynamic mechanical allodynia and
hyperaesthesia: the ‘‘Paint-brush test’’. The dynamic me-
chanical allodynia observed in neuropathic patients was measured
as previously developed in our laboratory and described in
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qPCR assay was conducted with one candidate housekeeping gene
for normalisation (actin, beta: Actb). The following primers were
used: Gabbr2, 59-AAGACCTTTGAAACACTCTGCAC-39
(forward) and 59-CCAAAGGCAGTTGTGTAGCC-39 (reverse);
Actb, 59-CCCGCGAGTACAACCTTCT-39 (forward) and 59CGTCATCCATGGCGAACT-39 (reverse). qPCR was performed on all samples (saline-saline; saline-oxycodone; salinemorphine; vincristine-saline; vincristine-oxycodone; vincristinemorphine). All fluorescence data were processed using a postPCR data analysis software program (LightCycler relative
Quantification Software version 1.0; Roche), providing an
automatic calculation of a normalized ratio. Saline samples were
used for normalisation. The ratios of each gene of interest were
compared among samples, using an unpaired t-test.
receptor staining. As previously reported [25,26], we observed
staining in the superficial laminae (Figure S1B) and in deeper
laminae (Figure S1B). Spinal cord sections incubated without
primary antibody did not display any staining (data not shown).
GABAB2 receptor labelling in dorsal root ganglia
Immunostainings were performed on tissues from 4 animals per
group. Double immunofluorescent stainings for GABAB2 receptor
and bIII tubulin (a marker of all neuronal cells) were performed to
determine the percentage of neurons expressing GABAB2 receptors. Sections were incubated overnight at room temperature with
a rabbit anti-GABAB2 receptor antibody (1:300, ref. 52248,
Abcam, Cambridge, UK) and mouse anti-bIII Tubulin antibody
(1:2000, ref. G7121, Promega, San Luis Obispo, CA USA);
antibodies were diluted in PBS-T-azide (PBS 0.02 M containing
0.3% Triton X-100 and 0.02% sodium azide). Following several
washes in PBS, sections were incubated for 2 h at room
temperature in Alexa FluorH 488 anti-rabbit IgG and Alexa
FluorH 350 anti-mouse IgG (Invitrogen, California, USA), diluted
1:1000 in PBS-T. Following several washes in PBS, sections were
mounted in Vectashield medium (Vector Laboratories, Burlingame, USA). A similar procedure was performed for GABAB2
receptor-GABAB1 receptor double immunostaining, using the
anti-GABAB1 receptor antibody (1:500; mouse anti-GABAB1
receptor, ref. 55051, Abcam, Cambridge, UK) and Alexa FluorH
546 anti-mouse IgG.
Immunohistochemistry
Tissue preparation. Twelve rats receiving vincristine treatment and 12 others receiving saline treatment were used for the
immunohistochemistry experiment. At D15, each group was
divided into 3 sub-groups that received chronic injections of
morphine (n = 4 vincristine-morphine, n = 4 saline-morphine
treated rats), oxycodone (n = 4 vincristine-oxycodone, n = 4
saline-oxycodone treated rats) or saline (n = 4 vincristine-saline,
n = 4, saline-saline treated rats). Rats were deeply anaesthetized at
the end of analgesic treatment (D19) with a 25 mg/ml urethane
injection (1.5 g/kg i.p), and perfused transcardially with 200 ml of
0.9% NaCl, followed by 500 ml of 4% paraformaldehyde (PFA) in
0.1 M phosphate buffer (PB), pH 7.4. Tissues were dissected out
and post-fixed at 4uC in the same fixative, either overnight (spinal
cord) or for 2 hours (DRGs). All tissues were further cryoprotected
in 30% sucrose (in 0.1 M PB, pH 7.4) overnight at 4uC. DRG
sections (14 mm thick) were serially cut using a cryostat (HM550,
Microm Microtech). Sections were mounted directly onto a
Superfrost slide such that two series of six slides corresponding to
the whole ganglion could be obtained. Two slides per animal were
used for each labelling. Spinal cord sections (30 mm thick) were cut
using a cryostat and collected in phosphate buffer saline (PBS;
0.02 M) containing 0.02% sodium azide.
Quantification of bIII tubulin- and GABAB2 receptorpositive neurons. Black and white 256 grey level images were
acquired using a 40X objective on a Zeiss microscope equipped
with a Zeiss Axiocam MRm camera. All acquisitions were
performed using the same acquisition setup. Quantification of
the percentage of GABAB2 receptor-positive cells was performed
by counting both the number of GABAB2 and bIII tubulin
immunopositive cells in DRG. At least 200 cells were counted per
DRG (more than four or five sections per rat). An analysis of
staining intensity and size diameter distribution was performed
using Image J software (http://rsb.info.nih.gov/ij/). The background staining intensity was subtracted from each image, and
results were expressed as the mean intensity of immunoreactivity
6 SEM. The mean diameter, percentage of positively stained cells,
and mean intensity of GABAB2 staining in DRG neurons were
compared between all groups by one-way ANOVA using
SigmaStat software, followed by the Student Newman–Keuls test
to detect differences between different groups.
Characterisation of the rabbit anti-GABAB2 antibody
Western Blot. The anti-GABAB2 antibody was tested using
the western blot method. Briefly, a rat was deeply anaesthetized
with pentobarbital (60 mg/kg i.p.; Ceva Santé Animale, Libourne,
France) and then euthanised by decapitation. The cortex of a
control animal was homogenised in RIPA (radioimmunoprecipitation assay buffer; 50 mM Tris, pH 8, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate)
containing Complete protease inhibitor mixture (Complete,
Roche, Neuilly-sur-seine, France). The protein concentrations
were determined using a Biorad DC Protein Assay kit (Bio-Rad
Laboratories, California, USA). Spinal cord samples (30 mg of
proteins) were separated by 10% SDS-PAGE, and transferred to
nitrocellulose membranes. The membrane was then incubated
with the primary antibody rabbit anti-GABAB2 (ref. 52248,
Abcam, Cambridge, UK), diluted 1:1000 in 20 mM Tris,
pH 7.5, 500 mM NaCl, 0.1% Tween 20 (TBS-T). After several
washes in TBS-T, the membrane was incubated in an anti-rabbit
IgG HRP secondary antibody (1:10000) for 1 h at room
temperature. Following several washes in TBS-T, the membrane
was treated with the ECL-plus reagent (10 min) for detection by
autoradiography. A specific band was observed at 106-110 kDa,
corresponding to the GABAB2 receptor (Figure S1A).
Immunohistochemistry. Immunostainings performed with
this antibody on spinal cord revealed characteristic GABAB2
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GABAB2 receptor labelling in the spinal dorsal
horn. Free floating serial sections from the lumbar segment
L4-L5 were incubated in rabbit anti-GABAB2 receptor antibody
(1:300; ref. 52248, Abcam, Cambridge, UK). This was either
performed alone for quantification, or in combination with the
following antibodies for double or triple staining: 1) IB4, a FITCconjugated isolectine (1:700; Isolectin B4 FITC conjugate, ref. L
2895, Sigma-Aldrich, St. Louis, MO); 2) mouse anti-CGRP
(1:500; ref. C 9487, Sigma-Aldrich, St. Louis, MO); 3) guinea pig
anti-vGlut2 (1:5000; ref. AB2251, Chemicon, Temecula, CA
USA); or 4) mouse anti-GABAB1 receptor (1:500; ref. 55051,
Abcam, Cambridge, UK, [27]). Tissues were finally washed in
PBS, and mounted in Vectashield medium (Vector, Burlingame,
USA).
Quantification of GABAB2 receptor labelling in the spinal
dorsal horn. To quantify GABAB2 receptor staining in the
dorsal horn of all subject groups, black and white 256 grey level
images were acquired using a 40X objective on a Zeiss microscope
equipped with a Zeiss Axiocam MRm camera. All acquisitions
were performed using the same acquisition setup. A 100 mm 6
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Oxycodone and Pain Relief
treated rats; { p,0.05, {{ p,0.01, {{{ p,0.001: vincristine-oxycodone
treated rats vs. vincristine-saline treated rats; 11 p,0.01, 111 p,0.001:
vincristine-morphine treated rats vs. vincristine-saline treated rats. For
figure clarity, the statistical significance symbols for the vincristine vs.
saline groups are not shown.
doi:10.1371/journal.pone.0091297.g001
500 mm region of interest was placed on the dorsal horn to
measure fluorescence intensity (FI) plot profiles (gray levels, in
arbitrary units, with respect to the distance from the surface, in
micrometers). Statistical significance was determined with a
Student’s unpaired t test for specific depth point, or one way
ANOVA followed by a Student Newman–Keuls test for mean
fluorescent intensity analysed per lamina (for all statistical tests:
**p,0.01;*p,0.05).
As a final note, each type of immunostaining in the DRG or
dorsal horn was simultaneously performed for all animals, in order
to maintain consistency and allow a statistical analysis of all results.
Omission of the primary antibody, or any other stage in the
protocol, did not result in any labelling.
Results
Oxycodone maintained a strong analgesic effect after
chronic treatment as compared to morphine in
vincristine-treated rats
As previously observed [28], at the end of vincristine treatment
(D15), vincristine-treated rats displayed a significant increased
static (Figure 1B-C) and dynamic (Figure S2A–B) mechanical
sensitivity, as compared to the baseline (BL, i.e. beginning of the
treatment) and saline-treated rats. A single injection of morphine
or oxycodone was capable of totally reversing static mechanical
allodynia, hyperalgesia (D15 T30, Figures 1B and C, respectively)
and dynamic mechanical allodynia (D15 T30, Figure S2A). The
analgesic effect of oxycodone (measured by the Pinch test) was
more potent than the analgesic effect of morphine on static
mechanical hyperalgesia (D15 T30, Figure 1C). Moreover,
dynamic mechanical hyperesthesia (measured with the rough
paintbrush test) was only attenuated by morphine, whereas it was
totally reversed by oxycodone (D15 T30, Figure S2B). At the end
of the analgesic chronic treatment on day 19 (D19), only
oxycodone was able to maintain the analgesic effect observed at
D15 on mechanical sensitivity (D19 T30, Figure 1B-C and Figure
S2A–B). By this point, morphine had completely lost its analgesic
effect on dynamic mechanical sensitivity (Figure S2A–B).
Extensive analysis of gene expression modified by
chronic analgesic treatment
Figure 1. Time-course of static mechanical sensitivity. A:
Schematic representation of the experimental paradigm. Rats received
i.p. injections of vincristine (0.1 mg/kg/day) during 2 cycles of 5 days.
Analgesic treatment started at D15, and was performed for 5 days until
D19. The analgesic effect of oxycodone or morphine was tested on D15
and D19, with a time-course of 30, 60 and 90 minutes after injection.
These injections are illustrated by black triangles. At D15, all groups of
vincristine-treated rats presented static mechanical allodynia (B) and
hyperalgesia (C), as compared to control rats and the baseline (BL). B:
Static mechanical allodynia measured using the electronic Von Frey
test. The measured static mechanical allodynia was totally reversed after
a single injection of morphine or oxycodone, although only oxycodone
maintained a strong analgesic effect at D19. C: Static mechanical
hyperalgesia measured by the ‘‘Pinch Test’’. Oxycodone induced an
analgesic effect in vincristine-oxycodone treated rats after a single
injection, and maintained its analgesic effect at D19. All data are
expressed as mean 6 SEM. (n = 7 vincristine-morphine treated rats;
n = 6 saline-morphine treated rats), (n = 7 vincristine-oxycodone treated
rats; n = 6 saline-oxycodone treated rats), and (n = 7 vincristine-saline
treated rats; n = 6 saline-saline treated rats). *p,0.05, **p,0.01, ***p,
0.001: vincristine-oxycodone treated rats vs. vincristine-morphine
PLOS ONE | www.plosone.org
We conducted a large-scale DNA microarray study based on
pan-genomic chips, using Illumina technology on the dorsal root
ganglion (DRG). This was aimed at systematically detecting genes
whose expression was altered by chronic morphine or oxycodone
treatment in either saline- or vincristine-treated rats. Among the
22,517 probes included in the microarray, 757 were differentially
expressed by the end of analgesic treatment (D19), corresponding
to 316 genes (Table S1). The complete list of dysregulated genes is
catalogued in Table S1. Among these results, we report 145 genes
that are differentially expressed after analgesic treatment in control
rats (Table S1). This list could be applied to improve our
understanding of the molecular mechanisms underlying chronic
opioid treatment in steady state, and in particular the mechanisms
of tolerance observed after chronic morphine treatment.
In this study, we chose to investigate the molecular mechanisms
controlling the sustained analgesic effect of oxycodone in
5
March 2014 | Volume 9 | Issue 3 | e91297
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Table 1. List of genes differentially regulated between vincristine-morphine vs. vincristine-oxycodone treated animals.
ACCESSION
SYMBOL
V-Mor
V-Oxy
V-NaCl
S-Mor
S-Oxy
S-NaCl
Variance analysis Vin
vs Control
Mor vs Oxy Vincritsine
animals
Mor vs Oxy Control animals
p
p
p
diff
diff
NM_144737
Fmo2
7,19
7,67
7,27
7,01
7,03
7,01
0,01
0,04
20,48
0,92
20,02
NM_145670
Bcas1
8,88
9,22
8,95
9,16
8,81
9,15
0,77
0,01
20,34
0,01
0,35
NM_031345
Dsipi
11,74
12,02
11,78
11,86
11,83
11,71
0,52
0,04
20,28
0,82
0,03
NM_012829
Cck
6,26
6,53
6,29
6,34
6,31
6,31
0,48
0,01
20,27
0,79
0,03
NM_134398
P34
6,06
6,28
6,22
6,24
6,16
6,24
0,57
0,01
20,22
0,26
0,08
NM_053349
Sox11
6,02
6,24
6,09
6,12
6,12
6,05
0,8
0,02
20,22
0,98
0
NM_013066
Mtap2
6,16
6,39
6,33
6,29
6,18
6,35
0,73
0,03
20,22
0,27
0,11
0,1
NM_001004132
Pctk1
8,5
8,71
8,54
8,84
8,73
8,7
0,01
0,04
20,21
0,28
NM_203367
Zmynd11
6,2
6,4
6,48
6,43
6,34
6,41
0,5
0,02
20,2
0,28
0,09
NM_001006985
Mrpl13
10,81
11,01
10,97
10,97
10,92
10,92
0,95
0,02
20,2
0,46
0,06
6
NM_053555
Vamp5
8,05
8,25
8,18
8,15
8,13
8,12
0,62
0,03
20,2
0,85
0,02
NM_031613
Tmod2
7,74
7,94
7,82
7,81
7,82
7,93
0,68
0,04
20,19
0,93
20,01
NM_031802
Gabbr2
11,15
11,34
11,17
11,19
11,19
11,22
0,64
0,04
20,19
0,99
0
NM_012839
Cycs
12,27
12,44
12,4
12,33
12,22
12,33
0,11
0,04
20,18
0,2
0,11
NM_134351
Mat2a
6,27
6,45
6,4
6,36
6,19
6,39
0,11
0,01
20,18
0,01
0,17
0,21
Col5a2
9,93
10,1
10,12
10,14
9,93
10,18
0,45
0,04
20,17
0,02
Letm1
8,99
9,14
9,12
9,05
9,15
9,04
1
0,04
20,15
0,15
20,1
NM_080577
Nploc4
6,09
6,24
6,18
6,18
6,12
6,11
0,35
0,03
20,14
0,38
0,05
NM_053527
Cdc5l
9,2
9,34
9,34
9,38
9,18
9,32
0,98
0,03
20,14
0
0,2
NM_080586
Gabrg1
6,16
6,29
6,33
6,26
6,2
6,24
0,43
0,04
20,13
0,31
0,06
NM_017065
Gabrb3
5,95
6,07
6,01
6,07
6,05
6,06
0,08
0,01
20,12
0,59
0,02
NM_053483
Kpna2
9,33
9,45
9,41
9,4
9,49
9,41
0,28
0,04
20,12
0,1
20,1
NM_001004206
Pa2g4
6,03
5,92
5,98
6
5,99
5,96
0,8
0,01
0,1
NaN
NaN
NM_171993
Cdc20
6,43
6,32
6,35
6,38
6,55
6,44
0,01
0,04
0,11
0,01
20,17
20,11
NM_053854
Nat2
6,04
5,92
6,03
6,01
6,12
6,04
0,05
0,02
0,11
0,02
NM_031129
Tceb2
11,07
10,95
11
11,02
11,02
10,93
0,54
0,04
0,12
0,99
0
NM_021657
Phlpp
8,19
8,07
8,09
8,21
8,21
8,2
0,01
0,03
0,12
0,96
0
NM_133553
B3galt4
6,42
6,26
6,42
6,41
6,39
6,41
0,3
0,02
0,16
0,67
0,03
NM_058208
Socs2
6,33
6,13
6,1
6,12
6,19
6,23
0,85
0
0,2
0,24
20,07
NM_017030
Pccb
6,7
6,5
6,6
6,66
6,57
6,66
0,48
0,01
0,2
0,25
0,09
NM_001004259
Pnkp
6,6
6,39
6,51
6,52
6,55
6,46
0,78
0,01
0,21
0,63
20,03
NM_053719
Emb
8,22
8
8,16
8,03
8,11
8
0,15
0,03
0,22
0,38
20,09
NM_001007626
Ggps1
7,43
7,18
7,26
7,2
7,34
7,24
0,61
0,02
0,24
0,16
20,14
Oxycodone and Pain Relief
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XM_343564
NM_001005884
Data are illustrated as follows: 1) Gene accession number; 2) Gene symbol; 3) Mean expression of the genes for the six different groups; 4) results of variance analysis between vincristine and control: p-value; 5) results of Student’s
t-test between morphine and oxycodone in vincristine-treated rats: p-value and expression difference (morphine – oxycodone); 6) results of Student’s t-test between morphine and oxycodone in control rats: p-value and
expression difference (morphine – oxycodone).
doi:10.1371/journal.pone.0091297.t001
0,13
1,22
10,66
PLOS ONE | www.plosone.org
NM_031010
Alox15
9,43
10,22
6,43
6,29
6,38
0
0,01
0,74
20,01
21,27
1,12
8,77
NM_012947
Eef2k
7,65
8,67
7,65
8,92
9,18
0,48
0,04
0,03
0,01
0,92
0,91
0,28
0,3
0,04
0,03
0,44
0,19
6,6
6,45
6,51
6,72
6,73
6,5
6,57
6,76
6,51
6,21
6,94
Slc16a3
NM_001007612
NM_030834
Ccl7
6,66
p
diff
p
V-Mor
SYMBOL
ACCESSION
Table 1. Cont.
V-Oxy
V-NaCl
S-Mor
S-Oxy
S-NaCl
p
diff
Mor vs Oxy Control animals
Variance analysis Vin
vs Control
Mor vs Oxy Vincritsine
animals
Oxycodone and Pain Relief
neuropathic pain, as compared to morphine. Our analysis was
focused on genes differentially expressed after analgesic treatment
in vincristine-treated rats; we thus identified 37 genes (Table S1).
The difference in gene expression between vincristine-morphine
and vincristine-oxycodone conditions was calculated (Table 1),
revealing 15 genes whose expression was up-regulated after
morphine treatment; this can also be expressed as (vincristinemorphine gene expression) - (vincristine-oxycodone gene expression) .0. Furthermore, we identified 22 genes whose expression
was up-regulated after oxycodone treatment; this can also be
expressed as (vincristine-morphine gene expression) - (vincristineoxycodone gene expression) ,0 (Table 1). Among the genes upregulated after oxycodone treatment, we observed 3 genes that
regulate receptor activity, classified in the ‘Receptor’ subcategory.
Interestingly, these genes were: i) Gabbr2, coding for the GABAB2
receptor; and ii) Gabrb3 and Gabrg1, coding respectively for the
GABAA R subunit b3 and GABAA R subunit c1 (Table 2). The
differential expression of these three genes was significant between
morphine- and oxycodone-treated rats after vincristine treatment.
Although we could not exclude a role of the GABAA receptor in
chronic opioid treatment, as reported in previous studies [29,30],
we chose to concentrate the second part of this study on GABAB2
receptor expression, and their potential role in the long-lasting
analgesic effect of oxycodone on neuropathic pain.
Chronic oxycodone treatment induced an increase in
GABAB2 receptor expression in small diameter DRG
neurons, as well as their terminals in superficial laminae
of the dorsal horn
In order to validate our results, we verified the differential
expression of the GABAB2 receptor (Gabbr2) in the DRG of
vincristine-treated and control animals at the RNA and protein
levels. Using qPCR, we observed an up-regulation of Gabbr2
expression in vincristine-oxycodone treated animals as compared
to vincristine-morphine treated animals (1.9160.48 vs. 0.4760.24;
p,0.05) (Figure 2D). At the protein level, GABAB2 receptors were
exclusively expressed in small and medium diameter neurons in
DRGs (size #34 mm, Figures 2A-C and E). The size distribution
histogram of GABAB2 receptor immunoreactivity in DRG cells
was similar in all animal groups, with a peak at 24 mm (Figure 2E).
However, we observed an increase in the percentage of neurons
positive for GABAB2 staining in oxycodone-treated animals as
compared to morphine-treated animals, following vincristine
treatment (Figure 2F). Moreover, quantification of the mean
intensity of GABAB2 receptor staining showed a 53% increase in
vincristine-oxycodone treated rats as compared to vincristinemorphine treated rats, and an 84% increase compared to
vincristine-saline treated rats (Figure 2G).
In the spinal dorsal horn, the intensity of GABAB2 staining
exhibited a significant increase in both lamina I and the outer part
of lamina II (Figure 3A2D), with a 70% increase in lamina I only
in the vincristine-oxycodone treated group (Figure 3E). In
contrast, there was no significant change in the other groups, or
in deep laminae (Figure 3D).
Functionality and localisation of GABAB2 receptors
To be functional, GABAB receptors must be organised as
heterodimers comprising one GABAB1 and one GABAB2 subunit
(for review see [31]). Using double immunostaining, we effectively
observed their colocalisation in small and medium sized DRG
neurons (Figure S3A2C) and in superficial layers of the dorsal
horn (Figure S3D2F), suggesting that functional GABAB receptors
are expressed at these levels. In addition, we observed that
7
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Oxycodone and Pain Relief
Table 2. Modification of genes regulating GABA receptor activity expression after oxycodone treatment.
GABAA R subunit b3
GABAA R subunit c1
GABAB2 R
Mean Expression
Vincristine-morphine
5.9560.05
6.1660.10
11.1560.08
Vincristine-oxycodone
6.0760.09
6.2960.04
11.3460.12
Vincristine-saline
6.0160.02
6.3360.10
11.1760.13
Control-morphine
6.0760.03
6.2660.07
11.1960.12
Control-oxycodone
6.0560.02
6.2060.11
11.1960.14
Control-saline
6.0660.09
6.2460.03
11.2260.16
p-value vincristine vs. control
0.081
0.429
0.644
Difference of expression
20.048
0.027
0.024
p-value morphine vs. oxycodone in vincristine animals
0.012
0.038
0.047
Difference of expression
20.118
20.129
20.192
p-value morphine vs. oxycodone in control animals
0.591
0.312
0.992
Difference of expression
0.023
0.060
20.001
Statistics
Data are illustrated as follows: 1) Mean expression value for all groups; 2) Statistical analysis: results of variance analysis between vincristine and control: p-value and
expression difference (vincristine – control); results of Student’s t test between morphine and oxycodone in vincristine-treated rats: p-value and expression difference
(morphine – oxycodone); results of Student’s t test between morphine and oxycodone in control rats: p-value and expression difference (morphine – oxycodone). The
three genes regulating GABA receptor activity expression were exclusively modified after analgesic treatment in vincristine-treated animals.
doi:10.1371/journal.pone.0091297.t002
Figure 2. Up-regulation of GABAB2 receptor expression in small diameter DRG neurons after chronic oxycodone treatment. A2C:
Immunofluorescent staining showing GABAB2 receptor immunoreactive neurons (arrowheads) in representative vincristine-saline (A), vincristinemorphine, (B) and vincristine-oxycodone (C) treated animals. Scale bars = 50 mm. D: Relative expression of Gabbr2 in the DRG of vincristine-treated
rats determined by qPCR. The results validate the up-regulation of the Gabbr2 gene observed by DNA microarray in the vincristine-oxycodone
treated rat, as compared to a vincristine-morphine treated rat. Data obtained from vincristine-treated rats were normalized to control rat data (n = 4
per group; *p,0.05 between vincristine-oxycodone treated rats and vincristine-morphine treated rats). Data were expressed as mean 6 SEM. E:
Histogram showing the size distribution frequency of GABAB2 receptor expression in the DRG for all vincristine groups. Note that the size distribution
frequency was identical in the three groups studied. F: Percentage of GABAB2 receptor-positive neurons in the three animal groups. Oxycodone
induced an increased percentage of GABAB2 receptor-positive neurons as compared to morphine treatment. G: Analysis of the mean intensity of
GABAB2 receptor staining in the three vincristine-treated animals. Oxycodone induced a 53% increase in GABAB2 receptor staining in vincristineoxycodone treated rats as compared to vincristine-morphine treated rats, and an 84% increase as compared to vincristine-saline treated rats. (n = 4
vincristine-morphine treated rats, n = 4 vincristine-oxycodone treated rats, and n = 4 vincristine-saline treated rats) *p,0.05 between vincristineoxycodone treated rats and vincristine-morphine treated rats; #p,0.05 between vincristine-oxycodone treated rats and vincristine-saline treated
rats. Data are expressed as mean 6 SEM.
doi:10.1371/journal.pone.0091297.g002
PLOS ONE | www.plosone.org
8
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Oxycodone and Pain Relief
Figure 3. Up-regulation of GABAB2 receptor expression in superficial laminae of the spinal cord after chronic oxycodone treatment.
A2C: Immunofluorescent staining showing GABAB2 receptor labelling in the dorsal horn in (A) vincristine-saline, (B) vincristine-morphine, and (C)
vincristine-oxycodone treated animals. D: Analysis of the fluorescence intensity profiles of GABAB2 receptor immunoreactivity from superficial to
deeper layers in control/vincristine treated animals chronically treated with saline, morphine or oxycodone. The selected zone was a vertical area in
laminae I through IV (white selection in A). The insert shows the mean fluorescent intensity 6 SEM in the three groups of vincristine-treated rats as
mean 6 SEM. For ease of reading, it was not possible to show the SEM of all groups. E: Mean fluorescent intensity of GABAB2 receptor
immonoreactivity at the level of the first 50 mm of the spinal cord dorsal horn. Vincristine treatment did not alter the intensity of GABAB2 receptor
staining as compared to saline-treated animals. However, oxycodone (but not morphine) significantly increased the intensity of GABAB2 receptor
staining in the superficial lamina. (n = 4 vincristine-morphine treated rats; n = 4 saline-morphine treated rats), (n = 4 vincristine-oxycodone treated rats;
n = 4 saline-oxycodone treated rats), and (n = 4 vincristine-saline treated rats; n = 4 saline-saline treated rats) *p,0.05 and **p,0.01 vincristineoxycodone vs. vincristine-morphine. Data are expressed as mean 6 SEM.
doi:10.1371/journal.pone.0091297.g003
GABAB2 receptors mainly colocalised within peptidergic CGRPpositive fibres (Figure S4A2C; Figure 4B2C2D, crossed arrows),
and not in non-peptidergic IB4-positive terminals (Figure S4D2
F). Notably, we observed that GABAB2 receptors were present in
glutamatergic presynaptic terminals (vGlut2 positive) containing
CGRP (Figure 4A2D, arrows).
We directly examined the role of GABAB receptors in the longlasting analgesic effect of oxycodone by assessing the effect of
intrathecal administration of saclofen (10 mg), a selective GABAB
receptor antagonist [21,22] (Figure 5A). The relieving effect of
oxycodone on static mechanical allodynia was totally blocked by
the antagonist (Figure 5B), whereas its analgesic effect on
mechanical hyperalgesia was only partially blocked (Figure 5C).
Interestingly, intrathecal administration of saclofen had no effect
on the analgesic action of oxycodone after a single injection at D15
(Figure S5A2B). Moreover, in order to demonstrate the specific
involvement of up-regulated GABAB receptor function in the longlasting analgesic effect of chronic oxycodone injection, we assessed
the effect of an intrathecal administration of saclofen after a single
injection (D15) and after chronic administration of morphine
(D19). These results indicate that saclofen does not inhibit the
effect of acute or chronic treatment of morphine on mechanical
sensitivity in vincristine-treated rats (Figure S6A2B).
PLOS ONE | www.plosone.org
Discussion
The aim of this study was two-fold: to better understand the
mechanisms underlying the analgesic effect of both oxycodone and
morphine; and to compare these mechanisms of action in a model
of neuropathic pain induced by vincristine. Our results demonstrate that a large number of genes are dysregulated after opioid
analgesic treatment in vincristine-treated animals. These findings
also suggest an increased GABAergic tone in the superficial dorsal
horn induced by oxycodone treatment that is mediated by
GABAB2 receptor; this would explain the enhanced alleviation
of chronic neuropathic pain that we observed.
Attenuation of the analgesic effect of morphine
Vincristine-treated rats displayed all of the behavioural characteristics of peripheral sensory neuropathy observed in this model,
i.e. static and dynamic mechanical allodynia and hyperalgesia
[15,17,24,32]. As previously described in both patients and animal
models [2,3,5,8,33235], we observed that administration of
oxycodone or morphine alleviated symptoms of chronic neuropathic pain. However, repeated morphine injections lead to an
attenuation of its analgesic effect, while oxycodone maintained a
strong analgesic effect even after multiple injections. These
observations are consistent with previous reports demonstrating
morphine’s inefficacy in the symptomatic relief of neuropathic
9
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Oxycodone and Pain Relief
chronic treatment of morphine leads to a decrease in GABAergic
tonus [40,41]. Our DNA microarray analysis confirms this downregulation of GABAA receptor subunits (b3 and c1) in DRG
neurons induced by chronic morphine treatment. The functional
consequence of this decreased GABAA receptor synthesis in DRG
neurons could be a reduction in the spinal pre-synaptic
GABAergic tone, ultimately resulting in a persistent alteration of
synaptic signalling.
DNA microarray analysis as a tool to dissect molecular
mechanisms involved in opioid treatment
Our DNA microarray experiment was designed to address
distinct yet complementary questions. First, which genes are
differentially expressed by vincristine treatment at the end of the
chronic analgesic treatment? This analysis is important to
comprehending the mechanisms underlying the maintenance of
neuropathic pain symptoms associated with vincristine treatment.
Since our DNA microarray experiment was performed days after
the end of the treatment and several weeks after the development
of neuropathic pain [17,28], these genes are likely to be involved in
the maintenance (and not development) of neuropathic pain
symptoms. Second, which genes are differentially expressed in
control animals when they are chronically treated with oxycodone
or morphine? As stated above, chronic morphine treatment leads
to an attenuation of its analgesic effect and the development of
tolerance. Understanding the different targets of gene alteration
induced by morphine at a steady state is important to clarifying the
molecular mechanisms involved in the drug action and the
development of this tolerance. As a final point, analysing the genes
differentially expressed by oxycodone or morphine treatment in
vincristine-treated animals provides us with further insight into the
molecular mechanisms controlling the sustained analgesic effect of
opioids in the neuropathic pain state.
Although these points are all important to address, their
thorough pursuit was beyond the scope of this study. Future
studies that aim to inquire into these different questions should
greatly benefit from the complete database of dysregulated genes
that we report here.
Figure 4. The GABAB2 receptor is located in CGRP-positive
glutamatergic presynaptic terminals. Confocal images of triple
immunostainings, comprised of vGlut2 (A, red), GABAB2 receptor (B,
green) and CGRP (C, blue), in a vincristine-oxycodone treated animal. D
is a merge of images A2C. We observed GABAB2 receptor expression
in CGRP-immunoreactive glutamatergic presynaptic terminals (arrow).
Glutamatergic presynaptic terminals immunoreactive for GABAB2
receptor were also observed (arrowhead), as well as CGRP-positive
GABAB2 terminals (crossed arrow). Scale bar A-D = 5 mm.
doi:10.1371/journal.pone.0091297.g004
pain in STZ-induced diabetes models [36,37], as well as an
observed slower rate of tolerance to oxycodone-6-oxyme (an
oxycodone derivative compound) as compared to morphine in
naive animals [18]. While morphine and oxycodone are both mopioid receptor agonists, previous studies have shown that these
two opioids exhibit different efficiencies and distinct analgesic
profiles in various pain models, such as the bone cancer pain
model and the SNL model [33]. A more recent study from that
same group has shown that modification of the m-opioid receptor is
responsible for the distinct analgesic effect of oxycodone and
morphine, and that m-opioid receptor activation is less attenuated
by oxycodone than by morphine [38]. Although the authors of
that study dismiss the hypothesis that, in the bone cancer pain
model, a change in the number of m-opioid receptors or a change
in the m-opioid receptor binding affinity might be the underlying
mechanisms, these hypotheses remain valid in our model. Finally,
as proposed by Nakamura et al., the differential activation of the mopioid receptor by oxycodone or morphine might be due to the
mechanism that regulates GTP binding onto G proteins in the mreceptor [38]. These changes in m-opioid receptor activation might
produce subtle but functionally important variations in intracellular cascade signalling, possibly leading to a modified gene
expression that is observed after oxycodone treatment, but not
after morphine treatment.
Previous studies have reported that repeated opiate administration alters gene expression in different regions of the nervous
system in rodents [29,30,39], which may contribute to plastic
changes associated with tolerance. It has also been shown that
morphine treatment alters the expression of several receptors in
the amygdala, including the GABAA receptor [29]. Moreover,
PLOS ONE | www.plosone.org
The GABAB receptor is involved in the long-lasting
analgesic effect of oxycodone
The spinal cord is the site of both pro- and anti-nociceptive
modulations. Released from primary afferent fibres, Substance P
(SP), CGRP and glutamate exacerbate neuronal activity and have
a pro-nociceptive action. Conversely, activation of GABAB
receptors inhibits voltage-dependent calcium channels via Gprotein-coupled adenylate cyclase [42,43] and reduces the release
of excitatory amino acids and SP from primary afferent terminals
in the spinal cord [44,45]. Therefore, an increase in the
GABAergic tonus decreases neuronal excitation and has an antinociceptive property.
In vincristine-treated animals (but not in naive animals), chronic
treatment with oxycodone specifically induced an up-regulation of
GABAB2 receptor expression as compared to vincristine-morphine
treated rats. We also found that GABAB2 receptors were expressed
in superficial layers of the dorsal horn, particularly in glutamatergic presynaptic terminals containing CGRP. As previously
reported, the up-regulation of GABAB receptors in DRG neurons
leads to an increase in inhibitory transmission tonus and a
decrease in excitatory transmission tonus, which could explain the
long-lasting analgesic effect observed during chronic oxycodone
administration in neuropathic pain conditions. Our hypothesis is
that the up-regulation of GABAB2 receptor expression in CGRP
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Oxycodone and Pain Relief
hyperalgesia (C). (n = 6, vincristine-oxycodone-saclofen treated rats;
n = 6, vincristine-oxycodone-saline treated rats; n = 6 vincristine-salinesaclofen treated rats; n = 6, vincristine-saline-saline treated rats). ***p,
0.001: vincristine-oxycodone treated rats compared at different time
points; ## P,0.01, ### p,0.001: vincristine-oxycodone treated rats
vs. vincristine-saline treated rats; uup,0.01, uuup,0.001: vincristineoxycodone-saclofen treated rats vs. vincristine-oxycodone-saline treated rats; {{{ p,0.001: vincristine-oxycodone-saclofen treated rats vs.
vincristine-saline-saclofen treated rats.
doi:10.1371/journal.pone.0091297.g005
sensory afferents observed after chronic oxycodone treatment may
counteract the increased neuronal excitability induced by vincristine treatment. The inhibition of oxycodone’s analgesic effect
following an intrathecal injection of a selective GABAB receptor
antagonist confirms this hypothesis. Therefore, the alleviating
effect of oxycodone in neuropathic pain condition seems to involve
the spinal GABAergic tonus though GABAB receptors.
Although functional GABAB receptors must be comprised of a
heterodimer of GABAB1 and GABAB2 receptor subtypes to be
active [31], some studies report tissues in which the expression of
GABAB1 and GABAB2 receptors do not overlap. For example,
some tissues only express GABAB1 receptor subtypes (and not
GABAB2), suggesting a distinct role for GABAB1 in the absence of
GABAB2 [25]. In addition, independent maturation of GABAB1
and GABAB2 receptor subtypes has been observed during
postnatal development in the brain [46]. It has been demonstrated
that no biochemical or functional responses from the GABAB
receptor (or homomeric GBR1 or GBR2) are detectable in the
brain or retina of a GABAB1 -/- mouse mutant. [47251]. There is
a partial preservation of the GABAB2 receptor subtype staining in
the brain of GABAB1 -/- juvenile mice, suggesting a functional role
for GABAB2 receptor subtype proteins in the immature brain, in
addition to its contribution as a dimer in GABAB receptor
complexes[46]. Together, these data suggest that the GABAB1 and
GABAB2 receptor subtypes may play other roles, independent of
the presence of their partner. We could not exclude a different role
for the up-regulation of the GABAB2 receptor subtype in our
model.
In conclusion, this study provides evidence that treatment with
oxycodone produces a profound anti-nociceptive effect under a
neuropathic pain-like state. Furthermore, our results reveal a
thorough list of genes that are dysregulated in DRGs at the end of
chronic analgesic treatment. These findings will be indispensable
to future studies that aim to understand the analgesic effects of
oxycodone as compared to morphine in chemotherapy-induced
neuropathic pain.
Figure 5. The long-lasting analgesic effect of oxycodone is
reversed by i.t. injection of a GABAB antagonist. A: Schematic
representation of the experimental paradigm. Rats received i.p.
injection of vincristine (black horizontal bar). The oxycodone treatment
started at D15, while animals presented mechanical hyperalgesia (dark
grey horizontal bar). Behavioural tests, illustrated by black triangles,
were performed at D0 (BL), D15 and D19, before (T0) and 30 min after
(T30) oxycodone injection, to control for the analgesic effect of
oxycodone. The functional involvement of GABAB receptors in this longasting analgesic effect of oxycodone was tested by intrathecal (i.t.)
injection of saclofen (10 mg) versus saline injection (grey arrow), 4 hours
after the previous test (D19). Rats were injected again with oxycodone
or saline (D199). Fifteen minutes after this second injection, rats were
injected intrathecally with saclofen or saline and tested 15 minutes after
the i.t. injection (T309). B, C: Effect of saclofen intrathecal injection on
oxycodone’s long-lasting effect on static mechanical allodynia (B) and
static mechanical hyperalgesia (C). Results are expressed as mean d
withdrawal threshold 6 SEM. The d-withdrawal
threshold
was
ðT30{T0Þ
|100 at
calculated using individual values as follows:
T0
ðT300 {T0Þ
|100 at D199. The results confirm the analgesic
D19 and
T0
action of oxycodone (left panel, D19), and show that saclofen
completely reversed the analgesic action on mechanical allodynia (B),
while only partially reversing the effect on static mechanical
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Supporting Information
Figure S1 Anti-GABAB2 receptor antibody specificity. A:
Representative western blotting of one spinal cord lysate, using the
anti-GABAB2 receptor antibody. A single band was observed at
106–110 kDa, corresponding to the expected weight of the
GABAB2 receptor protein. B: Anti-GABAB2 immunostaining on
a spinal cord section. Representative example of GABAB2 staining
in the dorsal horn, located primarily in the superficial laminae
(arrowheads). Some staining was observed in deep laminae as well
(arrows). Spinal cord sections incubated without primary antibody
showed no staining (data not shown).
(TIF)
Figure S2 Time-course of dynamic mechanical sensitivity. At D15, rats in all vincristine-treated groups suffered from
dynamic mechanical allodynia (A) and hyperesthesia (B). A: Mean
number of positive responses to the smooth paint-brush test. The
11
March 2014 | Volume 9 | Issue 3 | e91297
Oxycodone and Pain Relief
measured dynamic mechanical allodynia was totally reversed after
a single injection of morphine or oxycodone, although only
oxycodone maintained an analgesic effect at the end of the chronic
analgesic treatment (D19). B: Mean number of positive responses
to the rough paint-brush test. Only oxycodone maintained an
analgesic effect at the end of the chronic analgesic treatment
(D19). All data are expressed as mean 6 SEM. (n = 7 vincristinemorphine treated rats; n = 6 saline-morphine treated rats), (n = 7
vincristine-oxycodone treated rats; n = 6 saline-oxycodone treated
rats), and (n = 7 vincristine-saline treated rats; n = 6 saline-saline
treated rats). *p,0.05: vincristine-oxycodone treated rats vs.
vincristine-morphine treated rats; { p,0.05, {{ p,0.01: vincristine-oxycodone treated rats vs. vincristine-saline treated rats; 1 p,
0.05: vincristine-morphine treated rats vs. vincristine-saline treated
rats. For figure clarity, the statistical significance symbols for the
vincristine vs. saline groups are not shown.
(TIF)
mechanical allodynia (A) and static mechanical hyperalgesia (B).
After i.t. injection of saclofen, the analgesic effect of a single
injection of morphine at D15 was not modified (ns: vincristinemorphine-saclofen treated rats vs. vincristine-morphine-saline
treated rats). The analgesic effect of morphine was decreased at
D19 as compared to D15 (as expected), and was not modified by
the i.t. injection of saclofen (ns). All data are expressed as mean 6
SEM. 1 p,0.05, 111 p,0.001: vincristine-morphine-saclofen
treated rats vs. vincristine-saline-saclofen treated rats; # p,0.05,
### p,0.001: vincristine-morphine-saline treated rats vs.
vincristine-saline-saline treated rats.
(TIF)
Table S1 Complete list of genes dysregulated at the end
of analgesic treatment. Data are illustrated as follows: 1) Gene
accession number; 2) Gene symbol; 3) Mean expression of the
genes for the six different groups; 4) ident: an identifying z-z-z for
the statistical analysis significance (z corresponding to the three
tests; z = 1 if the null hypothesis is rejected with a risk of 5% or
z = 0); 5) results of variance analysis between vincristine and
control: p-value; 6) results of Student’s t test between morphine
and oxycodone in vincristine-treated rats: p-value and expression
difference (morphine – oxycodone); 7) results of Student’s t test
between morphine and oxycodone in control rats: p-value and
expression difference (morphine – oxycodone). Peach highlight
corresponds to an identifying number of 1_1_1 i.e. with a
statistically significant difference between vincristine and control,
and morphine and oxycodone in vincristine-treated rats, and
between morphine and oxycodone in control rats. Yellow
highlight corresponds to an identifying number of 1_0_1 i.e. with
a statistically significant difference between vincristine and control,
and between morphine and oxycodone in control rats. Green
highlight corresponds to an identifying number of 1_0_0 i.e. with a
statistically significant difference between vincristine and control,
only. Purple highlight corresponds to an identifying number of
0_1_1 i.e. with a statistically significant difference between
morphine and oxycodone in vincristine-treated rats, and between
morphine and oxycodone in control rats. Blue highlight
corresponds to an identifying number of 0_1_0 i.e. with a
statistically significant difference between morphine and oxycodone in vincristine-treated rats, only. In this group, there was
significant differential expression of several genes corresponding to
the GABAA R subunit b3 (Gabrb3), the GABAA R subunit c1
(Gabrg1), and the GABAB2 receptor (Gabbr2). Dark blue highlight
corresponds to an identifying number of 0_0_1 i.e. with a
statistically significant difference between morphine and oxycodone in control rats, only.
(DOC)
Figure S3 Colocalisation between GABAB1 and GABAB2
receptors in sensory neurons. A–F: Double immunostaining
showing the colocalisation of GABAB1 receptor (A, D) with
GABAB2 receptor (D, E) in the DRG (A–C) and in the spinal
dorsal horn (D–F) of a representative vincristine-oxycodone
treated animal. C and F are merged images of A and B, and D
and E, respectively. The results indicate colocalisation of both
GABAB1 and GABAB2 in small DRG neurons (arrow in A, B and
C) and in the superficial spinal dorsal horn (arrowheads in the
magnification of D, E and F). A–C, scale bar = 30 mm; D–F, scale
bar = 150 mm.
(TIF)
Figure S4 The GABAB2 receptor and CGRP colocalise in
superficial laminae of the dorsal horn. A–C: Double
immunostaining showing CGRP (A, C, red)/GABAB2 receptor (B,
C, green) positive fibres in a vincristine-oxycodone treated animal.
C is a merged image of A and B, showing colocalisation in laminae
I and II. D–F: Double immunostaining of IB4 (D, F, green)/
GABAB2 receptor (E, F, red) indicates little colocalisation in a
vincristine-oxycodone treated animal. Colocalisation was observed
principally in laminae II ‘out’. F is a merge of D and E. Scale bar
= 100 mm.
(TIF)
Figure S5 The acute analgesic effect of oxycodone is not
reversed by i.t. injection of a GABAB antagonist. A, B:
Effect of intrathecal saclofen (10 mg) injection on the acute
analgesic effect of oxycodone on static mechanical allodynia (A)
and static mechanical hyperalgesia (B). After i.t. injection of
saclofen, the analgesic effect of a single injection of oxycodone at
D15 was not modified (ns: vincristine-oxycodone-saclofen treated
rats vs. vincristine-oxycodone-saline treated rats). All data are
expressed as mean 6 SEM. {{{ p,0.001: vincristine-oxycodonesaclofen treated rats vs. vincristine-saline-saclofen treated rats;
### p,0.001: vincristine-oxycodone-saline treated rats vs.
vincristine-saline-saline treated rats.
(TIF)
Acknowledgments
The authors thank Mundipharma laboratory for kindly providing
oxycodone for this study. We also thank Brandon Loveall of ‘Improvence’
for English proofreading of the manuscript.
Author Contributions
The analgesic effect of morphine is not
modified by i.t. injection of a GABA B antagonist. A, B:
Effect of intrathecal saclofen (10 mg) injection on the acute (D15)
and chronic (D19) analgesic effect of morphine on static
Figure S6
Conceived and designed the experiments: KT BC SBM SP. Performed the
experiments: KT FM SD. Analyzed the data: KT IR. Wrote the paper:
KT SP.
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