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Cathepsin S in the spinal microglia facilitates morphine-induced antinociceptive tolerance in rats
Accepted Manuscript Title: Cathepsin S in the spinal microglia facilitates morphine-induced antinociceptive tolerance in rats Authors: Li Xiao, Xue Han, Xiao-e Wang, Qi Li, Yuan Chen, Yu Cui, Yu Chen PII: DOI: Reference:
S0304-3940(18)30719-5 https://doi.org/10.1016/j.neulet.2018.10.043 NSL 33898
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
31-7-2018 10-10-2018 18-10-2018
Please cite this article as: Xiao L, Han X, Wang X-e, Li Q, Chen Y, Cui Y, Chen Y, Cathepsin S in the spinal microglia facilitates morphineinduced antinociceptive tolerance in rats, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.10.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Cathepsin S in the spinal microglia facilitates morphine-induced antinociceptive tolerance in rats
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Authors:
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Li Xiaoa,1, Xue Hanb,1, Xiao-e Wanga, Qi Lia, Yuan Chenc, Yu Cuid,*, Yu Chena,**.
Affiliations:
Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University,
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a
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Guangzhou, 510000, China b
Neurobiology Research Center, Zhongshan School of Medicine, Sun Yat-sen
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c
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Guangzhou, 510317, China
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Department of Anesthesiology, Guangdong Second Provincial General Hospital,
University, Guangzhou 510080, China Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University,
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d
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Guangzhou, 510080, China
*Corresponding
author:
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Yu Cui, *mailing address: Department of Physiology, Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan 2 Rd, Guangzhou, 510080, China. Yu Chen, **mailing address: Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan 2 Rd, Guangzhou, 510000, China.
Telephone and fax numbers: 020-84113181 (Y. Cui), 020-87755766 (Y. Chen).
Email: [email protected] (Y. Cui), [email protected] (Y. Chen). 1
These authors contributed equally to this work.
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Abstract Microglia-derived lysosomal cysteine protease cathepsin S (CatS) is increasingly recognized as important mediators to exaggerate nociceptive signaling. However, the
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patterns and functional roles of CatS in morphine tolerance have never been
investigated. Here, we showed that mature form of CatS was exclusively upregulated in the spinal microglia following chronic morphine treatment. Pharmacological
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blockade of CatS before each morphine treatment prolonged the efficacy of morphine
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analgesia. Correspondingly, inhibition of CatS suppressed activation of spinal
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microglia and phosphorylated p38 MAPK. Finally, intrathecal injection of selective microglia inhibitor minocycline reduced upregulation of mature CatS induced by
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chronic morphine treatment. Our data provide novel insight into the cellular
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mechanisms underlying morphine antinociceptive tolerance and highlight CatS as a
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therapeutic target for preventing morphine tolerance.
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Keywords: morphine tolerance, Cathepsin S, microglia, p38 MAPK, minocycline
1. Introduction
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Opioids are the most widely used analgesics for severe pain. However, chronic
opioid use is limited by the development of opioid tolerance [1]. Therefore, better comprehension of the underlying mechanisms should facilitate the development of novel strategy for long-term use of morphine. Accumulating evidence suggests that neuron-microglia interaction at spinal level contributes to morphine antinociceptive tolerance [2]. Direct inhibition of microglia
activation [3-7] or blocking various neuronal signals that modulate microglial activity [6, 8, 9] attenuates morphine tolerance. Cathepsin S (CatS), a lysosomal cysteine protease expressed in microglia, has been implicated as an important player in neuron-microglia interaction [10]. CatS is synthesized as an inactive zymogen and proteolytically processed into mature form following removal of pro-peptide [11]. Once released, CatS cleaved the neuronal membrane-bound chemokine CX3CL1 [12],
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which acts exclusively at CX3CR1 receptor expressed by microglia and thereby activates microglia [13]. Contribution of microglial CatS to enhanced nociceptive
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signaling has been demonstrated in several experimental pain models [14-18]. Recently, we found that microglial CatS facilitates remifentanil-induced acute hyperalgesia [19]. However, its functional role in antinociceptive tolerance induced
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by chronic morphine remains unknown.
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In the present study, we aim to investigate the functional role of CatS in
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morphine tolerance by investigating its expression patterns in the spinal cord, as well
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2. Materials and Methods
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as effect of CatS inhibitor on microglial activation and morphine tolerance.
2.1. Ethics statement
All experimental procedures were approved by the Animal Care and Use
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Committee of Zhongshan School of Medicine of Sun Yat-sen University (Permit Numbers: SYXK (Guangdong) 2010–0107) and performed in accordance with the
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guideline of National Institutes of Health on the animal care and ethics. 2.2. Experimental animals Adult Sprague-Dawley (SD) rats (male; 260 ± 10 g; Sun Yat-Sen University
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Experimental Animal Center) were caged individually in a temperature-controlled room (24 ± 1°C) with a 12-h light-dark cycle (lights on at 08:00 am) and allowed free access to food and water. Animals would habituate for at least one week before any experimental procedures. 2.3. Intrathecal catheter implantation and drug administration Rats were implanted with intrathecal (i.t.) catheters according to our previous
study [3]. Under sodium pentobarbital (50 mg/kg, intraperitoneal) anesthesia, a polyethylene tube (PE10, 7 cm) was inserted intrathecally through the gap between vertebrae L5 and L6 to reach the spinal lumbar enlargement level. The catheter was fixed and the wound was closed in two layers with 4-0 polyester suture. All animals were allowed to recover for 7 days before other procedures. Drugs were administered in a volume of 10 µl followed by an additional 10 µl saline to flush the i.t. catheter.
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Morphine hydrochloride (Qinghai Pharmaceutical Factory, China) and minocycline
hydrochloride (Sigma) were dissolved in saline. The CatS inhibitor LHVS (NeoMPS,
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San Diego, USA) was solubilized in 20% Cremophor EL/saline. Morphine tolerance was induced by i.t. administration of morphine (15 μg, daily) for 7 days as described previously [20]. To investigate the effect of LHVS and minocycline on morphine
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tolerance, LHVS (10 or 50 nmol), vehicle (20% Cremophor EL/saline) or
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minocycline (50 μg) was i.t. given 30 min before each morphine administration.
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Dosages and concentration of drugs were chosen on the basis of previous studies [4,
2.4. Behavioral testing
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16, 20].
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Thermal and mechanical paw-withdrawal tests, as described previously [19, 21], were performed both before and 30 min after morphine injection every other day starting from day 1 (9:00 am - 12:00 am). For testing paw withdrawal thermal latency
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(PWTL), a radiant heat source (Series 8, Model 390G, IITC Life Science, USA) focus on the plantar skin of the hind paw through a transparent glass. The baseline latency
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was set to 4-6 s and a cut-off time of 15 s was set to avoid tissue damage. For testing paw withdrawal mechanical threshold (PWMT), the electronic Von Frey filaments (Almemo 2450, Anesthesiometer IITC, Inc., Woodland Hills, CA) was applied on the
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plantar hind paw with sufficient pressure to elicit a positive reaction. A cut-off of 80 g was chosen to avoid tissue damage. A positive pain reaction was defined by sudden paw withdrawal, paw flinching or licking following thermal or mechanical stimulation. Each rat was tested three times with an interval of 2 min and a change of right/left side. The mean of value was taken as the final latency or threshold of each rat. The percentage of maximal possible antinociceptive effect (%MPE) was
calculated by the formula: %MPE = [(Test value − Baseline) / (Cut-off value − Baseline)] × 100. 2.5. Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with cold saline followed by 4% paraformaldehyde. Spinal lumbar enlargements were dissected out and post-fixed in paraformaldehyde for 2 h and then
μm)
in
a
cryostat
and
mounted
serially
onto
microscope
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transferred into 30% sucrose for 48 h at 4°C. Subsequently, tissues were sectioned (20 slides
for
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immunohistochemistry. Sections were blocked with 5% donkey serum containing 0.3% TritonX-100 for 1 h at room temperature and then incubated with the primary antibodies against CatS (1:200, Santa Cruz, sc-6505, USA), GFAP (1:200, Millipore,
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MAB360, USA), OX-42 (1:200, Abcam, ab1211, USA) or NeuN (1:100, Abcam,
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ab104224) over night at 4°C, followed by the FITC- or Cy3-conjugated secondary
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antibody (1:400; Jackson ImmunoResearch, 715096151 or 711165152, USA) for 2 h at room temperature. Images were captured by using a fluorescence microscope
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(Olympus BX51, Japan) with FITC filters. For quantification of CatS and OX-42
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immunofluorescence, five spinal cord tissue sections were randomly selected from each rat (n = 6/group) and the intensity of fluorescence was analyzed by Image J (NIH, USA) software.
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2.6. Western blotting
Rats were anesthetized by overdose of sodium pentobarbital. Lumbar spinal
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dorsal horn (L4–L6) were harvested rapidly and sonicated on ice in a lysis buffer containing a cocktail of protease and phosphatase inhibitors. Tissue lysates were centrifuged at 13,000 rpm for 15 min at 4°C and supernatants were collected. Protein
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concentration was determined using a Coomassie plus protein assay kit (Thermo Scientific, USA). Protein samples were loaded and separated on SDS–PAGE gels and electroblotted onto PVDF membrane (Millipore) at 100 V for 90 min. The membranes were blocked with TBST containing 5% nonfat milk or BSA for 1 h at room temperature and then incubated with one of primary antibodies against CatS (1:200, Santa Cruz, sc-6505), Iba1 (1:1000, Abcam, ab15690), phosphorylated p38 (p-p38)
(1:1000, Cell Signaling Technology, 4511, USA), total p38 MAPK (1:1000, Cell Signaling Technology, 8690) overnight at 4°C. Membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody (1:10000, Jackson ImmunoResearch) for 1 h at room temperature before the blots were visualized in ECL solution (Pierce, USA) and exposed onto X-films. GAPDH was used as the loading control and detected using a corresponding primary
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antibody (1:10000, Sigma). Protein bands were quantified using a model GS-700 Imaging Densitometer (BioRad Laboratories, Italy) and analyzed by Image J
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software. Quantification analyses were expressed as the ratio of target proteins to their total proteins or loading control proteins. 2.7. Experimental design
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This study consisted of three phases.
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In phase 1, rats were randomly divided into six groups (n = 12): naïve, saline, and 1, 3, 5 or 7 days of morphine i.t. infusion. To evaluate morphine tolerance, rats
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were intrathecally administered saline (10 μl) or morphine (15 μg/10 μl) for 7 days.
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Paw withdrawal thermal latency (PWTL) (n = 6) and paw withdrawal mechanical
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threshold (PWMT) (n = 6) were tested before and 30 min after morphine infusion on days 1, 3, 5 and 7. For the time course analysis of CatS expression, rats receiving saline for 7 days or morphine infusion for 1, 3, 5 or 7 days were euthanized on
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respective days for immunohistochemical (n = 6) and western blot (n = 6) analysis. All the experiments were performed in double-blind manner.
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In phase 2, to explore the effect of CatS inhibitor LHVS on morphine tolerance,
rats were randomly divided into six treatment groups (n = 12): morphine, vehicle, LHVS (50 nmol), coadministration of morphine with vehicle or LHVS (10 or 50 nmol)
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for 7 days. Behavioral tests were performed on days 1, 3, 5 and 7. To investigate the effects of LHVS on microglial activation and CatS expression, rats from four groups (n = 12) (receiving vehicle, morphine, morphine plus vehicle and morphine plus LHVS (50 nmol) for consecutive 7 days) were euthanized on day 7 for immunohistochemical (n = 6) and Western blotting analysis (n = 6). In phase 3, to explore whether minocycline exhibits inhibitory effect on
CatS expression during morphine tolerance, rats were randomly divided into four groups (n = 12): saline, morphine, minocycline (50 μg) alone and morphine plus minocycline (50 μg). Behavioral tests were performed on days 1, 3, 5 and 7 and rats were euthanized on day 7 for immunohistochemical and western blotting analysis. 2.8. Statistical analysis All data were expressed as mean ± SEM. Repeated-measures ANOVA was used
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for analyzing behavioral data. For analysis of immunohistochemistry and Western
blotting, differences between groups were compared by a one-way ANOVA followed
USA). The significance level was defined as p < 0.05.
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3. Results
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by Bonferroni’s test. Statistical analyses were performed with SPSS 21.0 (SPSS Inc.,
3.1. Mature CatS expression in spinal microglia is upregulated by chronic
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morphine
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Intrathecal administration of 15μg morphine for 7 consecutive days resulted in a
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progressive decrease in analgesic effect in thermal (morphine, F1, 10 = 2845.166, p < 0.001; time, F3, 30 = 389.078, p < 0.001; morphine × time, F3, 30 = 344.654, p < 0.001)
30 =
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and mechanical paw-withdrawal test (morphine, F1, 10 = 1189.855, p < 0.001; time, F3, 104.456, p < 0.001; morphine × time, F3, 30 = 95.392, p < 0.001), indicating the
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establishment of morphine tolerance (Fig. 1A, B). In parallel with behavior testing, immunohistochemistry results exhibited a time-dependent increase in CatS expression
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in the spinal dorsal horn, which began on day 3 and peaked on day 7 after morphine administration (Fig. 2A, B). Western blotting results further identified that expression of mature CatS, but not pre-pro- and pro-form of CatS, was significantly increased chronic
morphine
treatment
(Fig.
2C,
D).
Furthermore,
CatS
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following
immunostaining was exclusively colocalized with microglial marker OX-42, but not with GFAP (astrocytic marker) or NeuN (neuronal marker) (Fig. 2E). 3.2. CatS inhibitor suppresses development of morphine tolerance To investigate the role of microglial CatS in morphine tolerance, irreversible CatS inhibitor LHVS was intrathecally administrated before each morphine treatment.
LHVS significantly delayed the reduction in morphine analgesia in both PWTL (treatment, F5, 30 = 465.815, p < 0.001; time, F3, 90 = 563.056, p < 0.001; treatment × time, F15, 90 = 73.257, p < 0.001) and PWMT (treatment, F5, 30 = 334.972, p < 0.001; time, F2.407, 72.219 = 450.615, p < 0.001; treatment × time, F12.036, 72.219 = 61.337, p < 0.001). Post hoc tests showed that intrathecal LHVS dose-dependently retained
microglial CatS was involved in the development of morphine tolerance.
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morphine analgesia on day 7 (p<0.001) (Fig. 3A, B). These data suggested that
3.3. CatS inhibitor suppresses morphine-induced microglial activation
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Endogenous CatS is responsible for microglial activation in neuropathic pain
model [16]. Here, we found that pretreatment with LHVS significantly suppressed morphine-induced microglial activation in the spinal cord (p<0.01) (Fig. 4A, B).
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Western blotting data further supported the inhibitory effect of LHVS on microglial
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activation (p<0.01) (Fig. 4C). Given that p38 MAPK was activated in spinal microglia
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following morphine treatment [3], we examined the effect of LHVS on phosphorylated level of p38 in the spinal cord. We found that intrathecal LHVS
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significantly inhibited p38 activation induced by chronic morphine (p<0.01) (Fig. 4D).
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These findings confirmed our hypothesis that CatS is responsible for the activation of spinal microglia, thereby contributing to the development of morphine tolerance. 3.4. Minocycline inhibits CatS upregulation and morphine tolerance
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Consistent with our previous study [4], pretreatment with intrathecal minocycline for consecutive 7 days attenuated morphine antinociceptive tolerance in both
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noxious thermal (treatment, F3, 20 = 612.335, p < 0.001; time, F3, 60 = 165.476, p < 0.001; treatment × time, F9, 60 = 67.683, p < 0.001) and mechanical test (treatment, F3, 20
= 480.607, p < 0.001; time, F3, 60 = 137.541, p < 0.001; treatment × time, F9, 60 =
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60.509, p < 0.001) (Fig. 5A, B). Minocycline alone had no significant effect on CatS expression compared with saline group (Fig. 5C, D). However, pretreatment with minocycline suppressed the increase in CatS immunoreactivity (p<0.001) (Fig. 5C). Western blotting further revealed that minocycline reduced mature CatS expression (p<0.001) (Fig. 5D). Although the protein level of both pre-pro- and pro- forms of CatS seemed to be lower in minocycline-pretreated group than those from morphine
group, the differences were not statistically significant.
4. Discussion In this study, we demonstrate that: (1) chronic morphine treatment induced upregulation of mature CatS in spinal microglia; (2) pretreatment with CatS inhibitor
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LHVS attenuated morphine tolerance, microglial activation and p38 MAPK phosphorylation induced by chronic morphine; (3) intrathecal minocycline reduced
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the maturation of CatS in morphine-tolerant rats. Together, our findings suggest that microglial CatS is a novel determinant in the development of morphine tolerance.
Microglia-derived lysosomal cathepsin S (CatS) is increasingly recognized as an
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important mediator to facilitate neuropathic and inflammatory pain [15-18]. Here, we found that chronic morphine induces a specific increase in mature form of CatS in
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spinal microglia, while leaving the pre-pro- and pro- form of CatS unaffected. The
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mechanisms responsible for morphine-induced maturation of CatS still remain largely
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unknown. It has been demonstrated that P2X7 receptor activation mediates release of active CatS from microglia following ATP stimulation [22]. Given that upregulation
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of microglial P2X7 receptor is induced by chronic intrathecal morphine and mediates morphine tolerance [9], activation of P2X7 receptor might be responsible for
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morphine-induced CatS maturation in our study. Recently, inhibition of neuronal reactive oxidative species (ROS) is shown to prevent CatS upregulation induced by
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acute use of opioid [19]. Evidence indicates that activation of mu-opioid receptor (MOR) constitutively expressed in microglia alters microglial activity [6, 23, 24], thus the possibility that MOR activation promotes CatS maturation in microglia cannot be
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excluded and warrants further investigation. CatS exerts its pronociceptive effects via cleavage of the neuronal CX3CL1 into
soluble active molecule [12, 15], which binds to its receptor CX3CR1 in microglia and activates p38 MAPK pathway [25]. Johnston and his colleagues reported that blockade of CX3CR1 signaling delayed the development of morphine tolerance [8]. Here, we found that intrathecal CatS inhibitor attenuates morphine tolerance.
Consistently, inhibition of CatS also suppressed microglial activation and phosphorylation of p38 MAPK induced by chronic morphine. Interestingly, Peng et al recently reported no significant changes in CX3CL1 expression at both protein and mRNA levels in the spinal cord following morphine treatment [26, 27]. It has been demonstrated that soluble CX3CL1 in the CSF, but not cell-associated CX3CL1 is increased following peripheral nerve injury [28]. Therefore, assay for the CX3CL1 in
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CSF may help to elucidate the mechanisms by which CatS mediates morphine tolerance. Several lines of evidence imply that CatS might be involved in morphine
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tolerance via other downstream effectors. Maturation of IL-1β, a critical inflammatory cytokines involved in morphine tolerance [29], can be promoted by CatS in cultured cortical microglia [22]. In addition, activation of proteinase-activated receptor 2
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(PAR2) [30] or specific Mas-related G protein–coupled receptors (Mrgpr) members
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[31, 32] have been shown to modulate morphine tolerance. Taken together, our
and contribute to morphine tolerance.
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findings suggest that endogenous CatS might be essential for microglial activation
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In addition, this study for the first time showed that intrathecal minocycline
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pretreatment exclusively suppressed morphine-induced maturation of CatS in the spinal microglia. Therefore, it might be possible that minocycline prevents the development of morphine tolerance via intervening CatS maturation and subsequently
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inhibiting microglial activation. Several lines of evidence have proposed that minocycline’s beneficial effects on morphine tolerance [4, 7, 33] are associated with
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inhibiting microglial activation and decreasing levels of pro-inflammatory cytokines. Interestingly, Posillico et al recently reported that gavage administration of minocycline (50 mg/kg) increased acute morphine analgesia and inhibited glial
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activation in male but not in female Sprague-Dawley rats [27]. Such discrepancy in the effect of minocycline on morphine analgesia might depend on dosage, administration routes and duration of treatment. Given that long-term treatment with minocycline is generally safe and well-tolerated in humans [34], clinical trials are necessary to evaluate its effect on morphine analgesia. Notably, a novel glial modulator, the endogenous fatty acid amide palmitoylethanolamide (PEA), was
recently demonstrated to be safe and exert antinociceptive effect in several animal models and clinical trials [35, 36]. Indeed, it has been reported that PEA significantly strengthened potency of morphine analgesia and attenuated the development of morphine tolerance [37, 38], suggesting the potential application of PEA as adjuvant to opioid-based therapy. 5. Conclusion
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These data offer novel mechanisms by which spinal microglia contributes to the
development of morphine tolerance and highlight that targeting CatS in the spinal
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microglia might be a valuable strategy to prolong the duration of opioid analgesics in chronic pain treatment. Founding sources:
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This work was supported by grants from Science and Technology Project in
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Guangzhou (No. 2012B031800368; No. 2009B030801110), the Fundamental
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Research Funds for the Central Universities (NO.12ykpy03) and Guangdong medical science and Technology Research Fund Project (NO.A2018013).
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Declarations of interest:
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The authors declare that no conflicts of interest exist.
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[28]
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Fig.1. Induction of morphine tolerance. Rats received daily intrathecal morphine or saline for 7
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days. Morphine antinociception was assessed using paw withdrawal thermal latency (PWTL) (A) and mechanical threshold (PWMT) (B). **p < 0.01, ***p < 0.001 versus saline group; n =
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6/group.
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Fig.2. Chronic morphine treatment induces upregulation of mature CatS in the spinal cord.
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(A, B) CatS immunoreactivity significantly increased in the spinal dorsal horn from rats receiving morphine treatment, compared with rats receiving saline treatment for 7 days. Scale bar: 200 μm.
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***p < 0.001 versus saline group; n = 6/group. (C, D) Western blotting analysis revealed that protein levels of mature form (MF), but not pre-pro form (PPF) and pro- form (PF), of CatS were
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upregulated in morphine group. ***p < 0.001 versus saline group; n = 6/group. (E) Double immunofluorescence staining showed that CatS immunoreactivity was predominantly colocalized
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with OX42 but not GFAP or NeuN. Scale bar: 50 μm.
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Fig.3. Effect of CatS inhibitor on morphine antinociceptive tolerance. Thermal (A) and mechanical (B) paw-withdrawal tests were performed on day 1, 3, 5 and 7. LHVS (10 or 50 nmol) intrathecally administrated 30 min before each morphine treatment delayed the development of
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antinociceptive tolerance. *p < 0.05, **p < 0.01, ***p < 0.001 versus morphine group, ###p <
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0.001 versus morphine plus LHVS (10 nmol) group; n = 6/group.
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Fig.4. Effect of CatS inhibitor on microglial activation induced by chronic morphine. (A, B)
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Immunohistochemical analysis showed that intrathecal LHVS (50 nmol) for 7 days suppressed chronic morphine-induced upregulation of OX-42 immunoreactivity in the spinal dorsal horn.
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Scale bar: 200 μm. ***p < 0.001 versus vehicle group, ##p < 0.01versus morphine group; n = 6/group. Western blotting analysis indicated that increased protein levels of Iba1 (C) and p-p38 (D) in the spinal cord induced by chronic morphine were significantly inhibited by LHVS. ***p <
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0.001 versus vehicle group, ##p < 0.01 versus morphine group; n = 6/group.
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Fig.5. Intrathecal minocycline attenuated morphine tolerance and upregulation of CatS. Intrathecal minocycline (50 μg) administrated 30 min before morphine treatment for consecutive 7
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days inhibited decreased morphine analgesia in thermal (A) and mechanical (B) paw-withdrawal tests. **p < 0.01, ***p < 0.001 versus morphine group; n = 6/group. (C) Immunohistochemical analysis showed that intrathecal minocycline prevented upregulation of CatS immunoreactivity on
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day 7. Scale bar: 200 μm. ***p < 0.001 versus saline group, ###p < 0.001 versus morphine group; n = 6/group. (D) Western blotting analysis showed that pretreatment with minocycline inhibited upregulated protein level of mature form (MF) of CatS on day 7. ***p < 0.001 versus saline group, ###p < 0.001 versus morphine group; n = 6/group.
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Fig.6. Schematic of proposed mechanism of morphine tolerance mediated by
CatS in the spinal cord. Consistent activation of μ-opioid receptor (MOR) or P2X7 receptor expressed on microglia promotes CatS maturation and subsequent release
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following chronic morphine exposure. Once released, CatS liberated CX3CL1 from
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neurons in the spinal dorsal horn. Then, CX3CL1 binds to CX3CR1 receptor on
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microglia and finally activated the p38 MAPK signaling pathway.
S0304-3940(18)30719-5 https://doi.org/10.1016/j.neulet.2018.10.043 NSL 33898
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
31-7-2018 10-10-2018 18-10-2018
Please cite this article as: Xiao L, Han X, Wang X-e, Li Q, Chen Y, Cui Y, Chen Y, Cathepsin S in the spinal microglia facilitates morphineinduced antinociceptive tolerance in rats, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.10.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Cathepsin S in the spinal microglia facilitates morphine-induced antinociceptive tolerance in rats
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Authors:
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Li Xiaoa,1, Xue Hanb,1, Xiao-e Wanga, Qi Lia, Yuan Chenc, Yu Cuid,*, Yu Chena,**.
Affiliations:
Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University,
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a
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Guangzhou, 510000, China b
Neurobiology Research Center, Zhongshan School of Medicine, Sun Yat-sen
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c
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Guangzhou, 510317, China
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Department of Anesthesiology, Guangdong Second Provincial General Hospital,
University, Guangzhou 510080, China Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University,
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d
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Guangzhou, 510080, China
*Corresponding
author:
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Yu Cui, *mailing address: Department of Physiology, Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan 2 Rd, Guangzhou, 510080, China. Yu Chen, **mailing address: Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan 2 Rd, Guangzhou, 510000, China.
Telephone and fax numbers: 020-84113181 (Y. Cui), 020-87755766 (Y. Chen).
Email: [email protected] (Y. Cui), [email protected] (Y. Chen). 1
These authors contributed equally to this work.
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Abstract Microglia-derived lysosomal cysteine protease cathepsin S (CatS) is increasingly recognized as important mediators to exaggerate nociceptive signaling. However, the
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patterns and functional roles of CatS in morphine tolerance have never been
investigated. Here, we showed that mature form of CatS was exclusively upregulated in the spinal microglia following chronic morphine treatment. Pharmacological
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blockade of CatS before each morphine treatment prolonged the efficacy of morphine
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analgesia. Correspondingly, inhibition of CatS suppressed activation of spinal
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microglia and phosphorylated p38 MAPK. Finally, intrathecal injection of selective microglia inhibitor minocycline reduced upregulation of mature CatS induced by
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chronic morphine treatment. Our data provide novel insight into the cellular
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mechanisms underlying morphine antinociceptive tolerance and highlight CatS as a
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therapeutic target for preventing morphine tolerance.
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Keywords: morphine tolerance, Cathepsin S, microglia, p38 MAPK, minocycline
1. Introduction
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Opioids are the most widely used analgesics for severe pain. However, chronic
opioid use is limited by the development of opioid tolerance [1]. Therefore, better comprehension of the underlying mechanisms should facilitate the development of novel strategy for long-term use of morphine. Accumulating evidence suggests that neuron-microglia interaction at spinal level contributes to morphine antinociceptive tolerance [2]. Direct inhibition of microglia
activation [3-7] or blocking various neuronal signals that modulate microglial activity [6, 8, 9] attenuates morphine tolerance. Cathepsin S (CatS), a lysosomal cysteine protease expressed in microglia, has been implicated as an important player in neuron-microglia interaction [10]. CatS is synthesized as an inactive zymogen and proteolytically processed into mature form following removal of pro-peptide [11]. Once released, CatS cleaved the neuronal membrane-bound chemokine CX3CL1 [12],
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which acts exclusively at CX3CR1 receptor expressed by microglia and thereby activates microglia [13]. Contribution of microglial CatS to enhanced nociceptive
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signaling has been demonstrated in several experimental pain models [14-18]. Recently, we found that microglial CatS facilitates remifentanil-induced acute hyperalgesia [19]. However, its functional role in antinociceptive tolerance induced
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by chronic morphine remains unknown.
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In the present study, we aim to investigate the functional role of CatS in
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morphine tolerance by investigating its expression patterns in the spinal cord, as well
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2. Materials and Methods
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as effect of CatS inhibitor on microglial activation and morphine tolerance.
2.1. Ethics statement
All experimental procedures were approved by the Animal Care and Use
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Committee of Zhongshan School of Medicine of Sun Yat-sen University (Permit Numbers: SYXK (Guangdong) 2010–0107) and performed in accordance with the
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guideline of National Institutes of Health on the animal care and ethics. 2.2. Experimental animals Adult Sprague-Dawley (SD) rats (male; 260 ± 10 g; Sun Yat-Sen University
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Experimental Animal Center) were caged individually in a temperature-controlled room (24 ± 1°C) with a 12-h light-dark cycle (lights on at 08:00 am) and allowed free access to food and water. Animals would habituate for at least one week before any experimental procedures. 2.3. Intrathecal catheter implantation and drug administration Rats were implanted with intrathecal (i.t.) catheters according to our previous
study [3]. Under sodium pentobarbital (50 mg/kg, intraperitoneal) anesthesia, a polyethylene tube (PE10, 7 cm) was inserted intrathecally through the gap between vertebrae L5 and L6 to reach the spinal lumbar enlargement level. The catheter was fixed and the wound was closed in two layers with 4-0 polyester suture. All animals were allowed to recover for 7 days before other procedures. Drugs were administered in a volume of 10 µl followed by an additional 10 µl saline to flush the i.t. catheter.
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Morphine hydrochloride (Qinghai Pharmaceutical Factory, China) and minocycline
hydrochloride (Sigma) were dissolved in saline. The CatS inhibitor LHVS (NeoMPS,
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San Diego, USA) was solubilized in 20% Cremophor EL/saline. Morphine tolerance was induced by i.t. administration of morphine (15 μg, daily) for 7 days as described previously [20]. To investigate the effect of LHVS and minocycline on morphine
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tolerance, LHVS (10 or 50 nmol), vehicle (20% Cremophor EL/saline) or
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minocycline (50 μg) was i.t. given 30 min before each morphine administration.
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Dosages and concentration of drugs were chosen on the basis of previous studies [4,
2.4. Behavioral testing
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16, 20].
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Thermal and mechanical paw-withdrawal tests, as described previously [19, 21], were performed both before and 30 min after morphine injection every other day starting from day 1 (9:00 am - 12:00 am). For testing paw withdrawal thermal latency
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(PWTL), a radiant heat source (Series 8, Model 390G, IITC Life Science, USA) focus on the plantar skin of the hind paw through a transparent glass. The baseline latency
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was set to 4-6 s and a cut-off time of 15 s was set to avoid tissue damage. For testing paw withdrawal mechanical threshold (PWMT), the electronic Von Frey filaments (Almemo 2450, Anesthesiometer IITC, Inc., Woodland Hills, CA) was applied on the
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plantar hind paw with sufficient pressure to elicit a positive reaction. A cut-off of 80 g was chosen to avoid tissue damage. A positive pain reaction was defined by sudden paw withdrawal, paw flinching or licking following thermal or mechanical stimulation. Each rat was tested three times with an interval of 2 min and a change of right/left side. The mean of value was taken as the final latency or threshold of each rat. The percentage of maximal possible antinociceptive effect (%MPE) was
calculated by the formula: %MPE = [(Test value − Baseline) / (Cut-off value − Baseline)] × 100. 2.5. Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with cold saline followed by 4% paraformaldehyde. Spinal lumbar enlargements were dissected out and post-fixed in paraformaldehyde for 2 h and then
μm)
in
a
cryostat
and
mounted
serially
onto
microscope
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transferred into 30% sucrose for 48 h at 4°C. Subsequently, tissues were sectioned (20 slides
for
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immunohistochemistry. Sections were blocked with 5% donkey serum containing 0.3% TritonX-100 for 1 h at room temperature and then incubated with the primary antibodies against CatS (1:200, Santa Cruz, sc-6505, USA), GFAP (1:200, Millipore,
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MAB360, USA), OX-42 (1:200, Abcam, ab1211, USA) or NeuN (1:100, Abcam,
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ab104224) over night at 4°C, followed by the FITC- or Cy3-conjugated secondary
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antibody (1:400; Jackson ImmunoResearch, 715096151 or 711165152, USA) for 2 h at room temperature. Images were captured by using a fluorescence microscope
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(Olympus BX51, Japan) with FITC filters. For quantification of CatS and OX-42
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immunofluorescence, five spinal cord tissue sections were randomly selected from each rat (n = 6/group) and the intensity of fluorescence was analyzed by Image J (NIH, USA) software.
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2.6. Western blotting
Rats were anesthetized by overdose of sodium pentobarbital. Lumbar spinal
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dorsal horn (L4–L6) were harvested rapidly and sonicated on ice in a lysis buffer containing a cocktail of protease and phosphatase inhibitors. Tissue lysates were centrifuged at 13,000 rpm for 15 min at 4°C and supernatants were collected. Protein
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concentration was determined using a Coomassie plus protein assay kit (Thermo Scientific, USA). Protein samples were loaded and separated on SDS–PAGE gels and electroblotted onto PVDF membrane (Millipore) at 100 V for 90 min. The membranes were blocked with TBST containing 5% nonfat milk or BSA for 1 h at room temperature and then incubated with one of primary antibodies against CatS (1:200, Santa Cruz, sc-6505), Iba1 (1:1000, Abcam, ab15690), phosphorylated p38 (p-p38)
(1:1000, Cell Signaling Technology, 4511, USA), total p38 MAPK (1:1000, Cell Signaling Technology, 8690) overnight at 4°C. Membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody (1:10000, Jackson ImmunoResearch) for 1 h at room temperature before the blots were visualized in ECL solution (Pierce, USA) and exposed onto X-films. GAPDH was used as the loading control and detected using a corresponding primary
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antibody (1:10000, Sigma). Protein bands were quantified using a model GS-700 Imaging Densitometer (BioRad Laboratories, Italy) and analyzed by Image J
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software. Quantification analyses were expressed as the ratio of target proteins to their total proteins or loading control proteins. 2.7. Experimental design
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This study consisted of three phases.
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In phase 1, rats were randomly divided into six groups (n = 12): naïve, saline, and 1, 3, 5 or 7 days of morphine i.t. infusion. To evaluate morphine tolerance, rats
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were intrathecally administered saline (10 μl) or morphine (15 μg/10 μl) for 7 days.
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Paw withdrawal thermal latency (PWTL) (n = 6) and paw withdrawal mechanical
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threshold (PWMT) (n = 6) were tested before and 30 min after morphine infusion on days 1, 3, 5 and 7. For the time course analysis of CatS expression, rats receiving saline for 7 days or morphine infusion for 1, 3, 5 or 7 days were euthanized on
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respective days for immunohistochemical (n = 6) and western blot (n = 6) analysis. All the experiments were performed in double-blind manner.
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In phase 2, to explore the effect of CatS inhibitor LHVS on morphine tolerance,
rats were randomly divided into six treatment groups (n = 12): morphine, vehicle, LHVS (50 nmol), coadministration of morphine with vehicle or LHVS (10 or 50 nmol)
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for 7 days. Behavioral tests were performed on days 1, 3, 5 and 7. To investigate the effects of LHVS on microglial activation and CatS expression, rats from four groups (n = 12) (receiving vehicle, morphine, morphine plus vehicle and morphine plus LHVS (50 nmol) for consecutive 7 days) were euthanized on day 7 for immunohistochemical (n = 6) and Western blotting analysis (n = 6). In phase 3, to explore whether minocycline exhibits inhibitory effect on
CatS expression during morphine tolerance, rats were randomly divided into four groups (n = 12): saline, morphine, minocycline (50 μg) alone and morphine plus minocycline (50 μg). Behavioral tests were performed on days 1, 3, 5 and 7 and rats were euthanized on day 7 for immunohistochemical and western blotting analysis. 2.8. Statistical analysis All data were expressed as mean ± SEM. Repeated-measures ANOVA was used
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for analyzing behavioral data. For analysis of immunohistochemistry and Western
blotting, differences between groups were compared by a one-way ANOVA followed
USA). The significance level was defined as p < 0.05.
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3. Results
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by Bonferroni’s test. Statistical analyses were performed with SPSS 21.0 (SPSS Inc.,
3.1. Mature CatS expression in spinal microglia is upregulated by chronic
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morphine
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Intrathecal administration of 15μg morphine for 7 consecutive days resulted in a
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progressive decrease in analgesic effect in thermal (morphine, F1, 10 = 2845.166, p < 0.001; time, F3, 30 = 389.078, p < 0.001; morphine × time, F3, 30 = 344.654, p < 0.001)
30 =
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and mechanical paw-withdrawal test (morphine, F1, 10 = 1189.855, p < 0.001; time, F3, 104.456, p < 0.001; morphine × time, F3, 30 = 95.392, p < 0.001), indicating the
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establishment of morphine tolerance (Fig. 1A, B). In parallel with behavior testing, immunohistochemistry results exhibited a time-dependent increase in CatS expression
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in the spinal dorsal horn, which began on day 3 and peaked on day 7 after morphine administration (Fig. 2A, B). Western blotting results further identified that expression of mature CatS, but not pre-pro- and pro-form of CatS, was significantly increased chronic
morphine
treatment
(Fig.
2C,
D).
Furthermore,
CatS
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following
immunostaining was exclusively colocalized with microglial marker OX-42, but not with GFAP (astrocytic marker) or NeuN (neuronal marker) (Fig. 2E). 3.2. CatS inhibitor suppresses development of morphine tolerance To investigate the role of microglial CatS in morphine tolerance, irreversible CatS inhibitor LHVS was intrathecally administrated before each morphine treatment.
LHVS significantly delayed the reduction in morphine analgesia in both PWTL (treatment, F5, 30 = 465.815, p < 0.001; time, F3, 90 = 563.056, p < 0.001; treatment × time, F15, 90 = 73.257, p < 0.001) and PWMT (treatment, F5, 30 = 334.972, p < 0.001; time, F2.407, 72.219 = 450.615, p < 0.001; treatment × time, F12.036, 72.219 = 61.337, p < 0.001). Post hoc tests showed that intrathecal LHVS dose-dependently retained
microglial CatS was involved in the development of morphine tolerance.
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morphine analgesia on day 7 (p<0.001) (Fig. 3A, B). These data suggested that
3.3. CatS inhibitor suppresses morphine-induced microglial activation
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Endogenous CatS is responsible for microglial activation in neuropathic pain
model [16]. Here, we found that pretreatment with LHVS significantly suppressed morphine-induced microglial activation in the spinal cord (p<0.01) (Fig. 4A, B).
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Western blotting data further supported the inhibitory effect of LHVS on microglial
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activation (p<0.01) (Fig. 4C). Given that p38 MAPK was activated in spinal microglia
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following morphine treatment [3], we examined the effect of LHVS on phosphorylated level of p38 in the spinal cord. We found that intrathecal LHVS
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significantly inhibited p38 activation induced by chronic morphine (p<0.01) (Fig. 4D).
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These findings confirmed our hypothesis that CatS is responsible for the activation of spinal microglia, thereby contributing to the development of morphine tolerance. 3.4. Minocycline inhibits CatS upregulation and morphine tolerance
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Consistent with our previous study [4], pretreatment with intrathecal minocycline for consecutive 7 days attenuated morphine antinociceptive tolerance in both
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noxious thermal (treatment, F3, 20 = 612.335, p < 0.001; time, F3, 60 = 165.476, p < 0.001; treatment × time, F9, 60 = 67.683, p < 0.001) and mechanical test (treatment, F3, 20
= 480.607, p < 0.001; time, F3, 60 = 137.541, p < 0.001; treatment × time, F9, 60 =
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60.509, p < 0.001) (Fig. 5A, B). Minocycline alone had no significant effect on CatS expression compared with saline group (Fig. 5C, D). However, pretreatment with minocycline suppressed the increase in CatS immunoreactivity (p<0.001) (Fig. 5C). Western blotting further revealed that minocycline reduced mature CatS expression (p<0.001) (Fig. 5D). Although the protein level of both pre-pro- and pro- forms of CatS seemed to be lower in minocycline-pretreated group than those from morphine
group, the differences were not statistically significant.
4. Discussion In this study, we demonstrate that: (1) chronic morphine treatment induced upregulation of mature CatS in spinal microglia; (2) pretreatment with CatS inhibitor
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LHVS attenuated morphine tolerance, microglial activation and p38 MAPK phosphorylation induced by chronic morphine; (3) intrathecal minocycline reduced
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the maturation of CatS in morphine-tolerant rats. Together, our findings suggest that microglial CatS is a novel determinant in the development of morphine tolerance.
Microglia-derived lysosomal cathepsin S (CatS) is increasingly recognized as an
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important mediator to facilitate neuropathic and inflammatory pain [15-18]. Here, we found that chronic morphine induces a specific increase in mature form of CatS in
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spinal microglia, while leaving the pre-pro- and pro- form of CatS unaffected. The
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mechanisms responsible for morphine-induced maturation of CatS still remain largely
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unknown. It has been demonstrated that P2X7 receptor activation mediates release of active CatS from microglia following ATP stimulation [22]. Given that upregulation
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of microglial P2X7 receptor is induced by chronic intrathecal morphine and mediates morphine tolerance [9], activation of P2X7 receptor might be responsible for
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morphine-induced CatS maturation in our study. Recently, inhibition of neuronal reactive oxidative species (ROS) is shown to prevent CatS upregulation induced by
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acute use of opioid [19]. Evidence indicates that activation of mu-opioid receptor (MOR) constitutively expressed in microglia alters microglial activity [6, 23, 24], thus the possibility that MOR activation promotes CatS maturation in microglia cannot be
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excluded and warrants further investigation. CatS exerts its pronociceptive effects via cleavage of the neuronal CX3CL1 into
soluble active molecule [12, 15], which binds to its receptor CX3CR1 in microglia and activates p38 MAPK pathway [25]. Johnston and his colleagues reported that blockade of CX3CR1 signaling delayed the development of morphine tolerance [8]. Here, we found that intrathecal CatS inhibitor attenuates morphine tolerance.
Consistently, inhibition of CatS also suppressed microglial activation and phosphorylation of p38 MAPK induced by chronic morphine. Interestingly, Peng et al recently reported no significant changes in CX3CL1 expression at both protein and mRNA levels in the spinal cord following morphine treatment [26, 27]. It has been demonstrated that soluble CX3CL1 in the CSF, but not cell-associated CX3CL1 is increased following peripheral nerve injury [28]. Therefore, assay for the CX3CL1 in
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CSF may help to elucidate the mechanisms by which CatS mediates morphine tolerance. Several lines of evidence imply that CatS might be involved in morphine
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tolerance via other downstream effectors. Maturation of IL-1β, a critical inflammatory cytokines involved in morphine tolerance [29], can be promoted by CatS in cultured cortical microglia [22]. In addition, activation of proteinase-activated receptor 2
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(PAR2) [30] or specific Mas-related G protein–coupled receptors (Mrgpr) members
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[31, 32] have been shown to modulate morphine tolerance. Taken together, our
and contribute to morphine tolerance.
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findings suggest that endogenous CatS might be essential for microglial activation
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In addition, this study for the first time showed that intrathecal minocycline
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pretreatment exclusively suppressed morphine-induced maturation of CatS in the spinal microglia. Therefore, it might be possible that minocycline prevents the development of morphine tolerance via intervening CatS maturation and subsequently
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inhibiting microglial activation. Several lines of evidence have proposed that minocycline’s beneficial effects on morphine tolerance [4, 7, 33] are associated with
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inhibiting microglial activation and decreasing levels of pro-inflammatory cytokines. Interestingly, Posillico et al recently reported that gavage administration of minocycline (50 mg/kg) increased acute morphine analgesia and inhibited glial
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activation in male but not in female Sprague-Dawley rats [27]. Such discrepancy in the effect of minocycline on morphine analgesia might depend on dosage, administration routes and duration of treatment. Given that long-term treatment with minocycline is generally safe and well-tolerated in humans [34], clinical trials are necessary to evaluate its effect on morphine analgesia. Notably, a novel glial modulator, the endogenous fatty acid amide palmitoylethanolamide (PEA), was
recently demonstrated to be safe and exert antinociceptive effect in several animal models and clinical trials [35, 36]. Indeed, it has been reported that PEA significantly strengthened potency of morphine analgesia and attenuated the development of morphine tolerance [37, 38], suggesting the potential application of PEA as adjuvant to opioid-based therapy. 5. Conclusion
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These data offer novel mechanisms by which spinal microglia contributes to the
development of morphine tolerance and highlight that targeting CatS in the spinal
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microglia might be a valuable strategy to prolong the duration of opioid analgesics in chronic pain treatment. Founding sources:
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This work was supported by grants from Science and Technology Project in
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Guangzhou (No. 2012B031800368; No. 2009B030801110), the Fundamental
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Research Funds for the Central Universities (NO.12ykpy03) and Guangdong medical science and Technology Research Fund Project (NO.A2018013).
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Declarations of interest:
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The authors declare that no conflicts of interest exist.
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Fig.1. Induction of morphine tolerance. Rats received daily intrathecal morphine or saline for 7
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days. Morphine antinociception was assessed using paw withdrawal thermal latency (PWTL) (A) and mechanical threshold (PWMT) (B). **p < 0.01, ***p < 0.001 versus saline group; n =
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6/group.
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Fig.2. Chronic morphine treatment induces upregulation of mature CatS in the spinal cord.
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(A, B) CatS immunoreactivity significantly increased in the spinal dorsal horn from rats receiving morphine treatment, compared with rats receiving saline treatment for 7 days. Scale bar: 200 μm.
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***p < 0.001 versus saline group; n = 6/group. (C, D) Western blotting analysis revealed that protein levels of mature form (MF), but not pre-pro form (PPF) and pro- form (PF), of CatS were
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upregulated in morphine group. ***p < 0.001 versus saline group; n = 6/group. (E) Double immunofluorescence staining showed that CatS immunoreactivity was predominantly colocalized
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with OX42 but not GFAP or NeuN. Scale bar: 50 μm.
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Fig.3. Effect of CatS inhibitor on morphine antinociceptive tolerance. Thermal (A) and mechanical (B) paw-withdrawal tests were performed on day 1, 3, 5 and 7. LHVS (10 or 50 nmol) intrathecally administrated 30 min before each morphine treatment delayed the development of
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antinociceptive tolerance. *p < 0.05, **p < 0.01, ***p < 0.001 versus morphine group, ###p <
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0.001 versus morphine plus LHVS (10 nmol) group; n = 6/group.
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Fig.4. Effect of CatS inhibitor on microglial activation induced by chronic morphine. (A, B)
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Immunohistochemical analysis showed that intrathecal LHVS (50 nmol) for 7 days suppressed chronic morphine-induced upregulation of OX-42 immunoreactivity in the spinal dorsal horn.
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Scale bar: 200 μm. ***p < 0.001 versus vehicle group, ##p < 0.01versus morphine group; n = 6/group. Western blotting analysis indicated that increased protein levels of Iba1 (C) and p-p38 (D) in the spinal cord induced by chronic morphine were significantly inhibited by LHVS. ***p <
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0.001 versus vehicle group, ##p < 0.01 versus morphine group; n = 6/group.
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Fig.5. Intrathecal minocycline attenuated morphine tolerance and upregulation of CatS. Intrathecal minocycline (50 μg) administrated 30 min before morphine treatment for consecutive 7
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days inhibited decreased morphine analgesia in thermal (A) and mechanical (B) paw-withdrawal tests. **p < 0.01, ***p < 0.001 versus morphine group; n = 6/group. (C) Immunohistochemical analysis showed that intrathecal minocycline prevented upregulation of CatS immunoreactivity on
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day 7. Scale bar: 200 μm. ***p < 0.001 versus saline group, ###p < 0.001 versus morphine group; n = 6/group. (D) Western blotting analysis showed that pretreatment with minocycline inhibited upregulated protein level of mature form (MF) of CatS on day 7. ***p < 0.001 versus saline group, ###p < 0.001 versus morphine group; n = 6/group.
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Fig.6. Schematic of proposed mechanism of morphine tolerance mediated by
CatS in the spinal cord. Consistent activation of μ-opioid receptor (MOR) or P2X7 receptor expressed on microglia promotes CatS maturation and subsequent release
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following chronic morphine exposure. Once released, CatS liberated CX3CL1 from
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neurons in the spinal dorsal horn. Then, CX3CL1 binds to CX3CR1 receptor on
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microglia and finally activated the p38 MAPK signaling pathway.