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Cathepsin S in the spinal microglia contributes to remifentanil-induced hyperalgesia in rats
Accepted Manuscript Cathepsin S in the spinal microglia contributes to remifentanil-induced hyperalgesia in rats L. Ye, L. Xiao, SY. Yang, JJ. Duan, Y. Chen, Y. Cui, Y. Chen PII: DOI: Reference:
S0306-4522(16)30730-8 http://dx.doi.org/10.1016/j.neuroscience.2016.12.030 NSC 17511
To appear in:
Neuroscience
Received Date: Revised Date: Accepted Date:
2 August 2016 13 December 2016 18 December 2016
Please cite this article as: L. Ye, L. Xiao, SY. Yang, JJ. Duan, Y. Chen, Y. Cui, Y. Chen, Cathepsin S in the spinal microglia contributes to remifentanil-induced hyperalgesia in rats, Neuroscience (2016), doi: http://dx.doi.org/ 10.1016/j.neuroscience.2016.12.030
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Title: Cathepsin S in the spinal microglia contributes to remifentanil-induced hyperalgesia in rats Running Head: ROS/CatS signaling involved in remifentanil-induced hyperalgesia Authors: L. Yea,1, L. Xiaoa,1, SY. Yanga, JJ. Duanb, Y. Chenb, Y. Cuic, *, Y. Chena, ** Institutions: a Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510000, China b Neurobiology Research Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510000, China c Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510000, China Corresponding author: * Department of Physiology, Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan 2 Rd, Guangzhou, 510080, China. ** Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan 2 Rd, Guangzhou, 510000, China Email: Email: [email protected] (Y Cui), [email protected] (Y Chen) 1
These authors contributed equally to this work
Category: Original article Founding sources: Science and Technology Project in Guangzhou (No. 2012B031800368; No. 2009B030801110), the Fundamental Research Funds for the Central Universities (NO.12ykpy03) Conflicts of interest: The authors have declared that no conflict of interest exists
Highlights 1. Remifentanil induces upregulation of matured cathepsin S (CatS) in spinal microglia 2. Contribution of CatS to remifentanil-induced hyperalgesia is linked to activation
of NMDA receptor 3. Minocycline inhibits CatS upregulation and hyperalgesia induced by remifentanil 4. Upregulation of matured CatS depends on ROS released from spinal neurons
ABSTRACT Cysteine protease Cathepsin S (CatS) expressed by spinal microglia has been shown to play a critical role in nerve injury and inflammation-induced chronic pain. However, whether microglial CatS contributes to remifentanil-induced acute hyperalgesia remains unstudied. In the present study, intravenous remifentanil infusion induced significant increase in the expression of premature and mature form of CatS in the activated microglia in the spinal cord. Spinal delivery of irreversible CatS inhibitor LHVS reduced hyperalgesia, attenuated activation of spinal microglia and blocked phosphorylation of NMDA receptor NR1 subunit induced by remifentanil. Furthermore, inhibition of microglia by minocycline effectively suppressed remifentanil-induced hyperalgesia, as well as CatS upregulation. In addition, remifentanil infusion also induced an increase in reactive oxygen species (ROS) levels in spinal neurons. Systemic administration of ROS scavenger PBN was sufficient to suppress remifentanil-induced painful hypersensitivity. Removal of ROS by PBN prevented upregulation of mature CatS in spinal microglia. However, increased protein level of premature form of CatS was not affected by PBN. Altogether, our findings demonstrate that neuronal ROS promote maturation of microglial CatS which facilitates activation of NMDA in the spinal dorsal horn. Therefore, such mechanism is involved in neuron-microglia positive feedback and contributes to remifentanil-induced hyperalgesia. Key words: remifentanil, hyperalgesia, Cathepsin S, microglia, minocycline, reactive oxygen species.
INTRODUCTION Remifentanil, a potent µ-opioid receptor agonist, is widely used for intraoperative anesthesia due to its rapid onset of action and clearance after withdrawal (Kim et al., 2014).
However,
hyperalgesia
may
develop
following
acute
remifentanil
administration and limits its potential clinical application (Joly et al., 2005, Bekhit, 2010). Although remifentanil-induced hyperalgesia has been reported in experimental studies and clinical observations in the past decade years (Hood et al., 2003), the mechanisms underlying such pathological process remain largely unknown. Growing evidence has suggested that spinal glial cells especially microglia modulate central sensitization (Watkins et al., 2001b, Obata et al., 2006) and thereby contributes to the development of opioid-induced tolerance (Cui et al., 2008) and hyperalgesia (Raghavendra et al., 2004, Ferrini and De Koninck, 2013). Recently, activation of spinal microglia and increased expression of cytokines are observed in the spinal cord following intraoperative remifentanil infusion (Romero et al., 2013, Sun et al., 2014). However, our understanding of the possible mechanism by which spinal microglia contributes to remifentanil-induced hyperalgesia is still limited. Cathepsin S (CatS) is a lysosomal cysteine protease expressed in macrophages, microglia and contributes to antigen presentation and adaptive immunity (Clark and Malcangio, 2012). CatS is initially synthesized as a pre-proenzyme and processed into its active mature form following removal of pro-peptide (Lecaille et al., 2002). Increased release of active CatS in spinal microglia has been detected in several neuropathic and inflammatory pain models (Clark et al., 2007, Clark et al., 2012, Nieto et al., 2016). Intriguingly, CatS has emerged as an important player in microglia-neuronal communication. It is found that selective activation of microglia by neuronal transmembrane fractalkine cleaved by CatS (Clark and Malcangio, 2012) contributes to increased pain sensitivity after morphine exposure (Johnston et al., 2004). Recently, fractalkine is found to induce the release of interleukin-1β (IL-1β) which modulates postsynaptic NMDA receptor signaling (Clark et al., 2015). Although activation of NMDA receptor in the spinal cord has been reported following remifentanil treatment (Zheng et al., 2012, Zhang et al., 2015, Ye et al., 2016),
whether neuron-microglia communication mediated by CatS-NMDA signaling in the spinal cord is involved in remifentanil-induced hyperalgesia has not been studied. The release of CatS from macrophage and microglia can be regulated by lipopolysaccharide (LPS) (Clark et al., 2009) and inflammatory cytokines (Liuzzo et al., 1999). The release of active CatS from microglia is also regulated by extracellular ATP via P2X7 receptor (Clark et al., 2010). Recently, regulation of CatS expression and activities by reactive oxygen species (ROS) has been reported in several cell lines (Cheng et al., 2008, Seo et al., 2009, Tsai et al., 2014). ROS are highly reactive metabolites of oxygen including superoxide, hydrogen peroxide and peroxynitrite (Dikalov and Harrison, 2014). Overproduction of ROS facilitates central sensitization and lead to hyperalgesia in several neuropathic pain models (Park et al., 2006, Gao et al., 2007, Lee et al., 2007, Kim et al., 2010). Of particular interest, it has been recently demonstrated that neuronal ROS is closely associated with the remifentanil-induced postoperative hyperalgesia (Zhang et al., 2014, Shu et al., 2015). Moreover, our recent report that ROS induces phosphorylation of NMDA receptor subunits following remifentanil treatment (Ye et al., 2016) further supports the role of ROS in remifentanil-induced
hyperalgesia.
Therefore,
whether
ROS
mediates
remifentanil-induced hyperalgesia via regulation of CatS in the spinal cord is worthy of investigation. In
the
present
study,
we
determined
whether
CatS
contributes
to
remifentanil-induced hyperalgesia via the mechanism associated with activation of NMDA signaling. Furthermore, we investigated regulation of CatS expression by neuronal ROS following remifentanil treatment in rats.
EXPERIMENTAL PROCEDURES Animals Male Sprague-Dawley rats (220-250g) were housed in a temperature-controlled room (22±1oC) with a 12-h light-dark cycle. All animals were acclimated for at least 1 week before any experimental procedures. All animal experimental procedures were approved by the Animal Care and Use Committee of Zhongshan school of Medicine,
and carried out in accordance with the guideline of National Institutes of Health on the animal care and the ethical guideline. Drug administration Remifentanil (Ren
Fu,
Co.,
Yichang,
China)
and
sevoflurane
(Maruishi
Pharmaceutical Co. Ltd, Japan) were supplied by the Department of Anesthesiology of the First Affiliated Hospital, Sun Yat-sen University (Guangzhou, China). Remifentanil was dissolved in saline (NaCl 0.9%) to a final concentration of 40 µg/ml. Following induction of anesthesia with sevoflurane (3.0% v/v) delivered via a nose mask, remifentanil were intravenous infused through tail vein over a period of 2 h (4 µg·kg−1·min−1) using an injection pump (Asena PK，UK). Control animals received the same volume of saline in identical conditions. ROS scavenger phenyl N-tert-butylnitrone (PBN; Sigma, St Louis, MO), CatS inhibitor LHVS (NeoMPS, San Diego, USA) or microglia inhibitor Minocycline （sigma，USA) was dissolved in saline. PBN (20 mg or 100 mg/kg) in total volume of 1 ml was injected intraperitoneally. LHVS (50 nmol/10 µl) or minocycline (50 µg/10 µl) was intrathecally injected as previous described (Mestre et al., 1994). In brief, the rats were anesthetized with sevoflurane and a 20 µl Hamilton microsyringe (GaoGe Co., Shanghai, China) was inserted between the L5 and L6 vertebrae. The entry of the needle into the subarachnoid space was confirmed when a sudden light tail flick occurred. Then LHVS or minocycline was injected into the subarachnoid space during a period of 30s, and the microsyringe was held for a further 10 s to prevent outflow of the agent. The inhibitor was given 30 min before remifentanil infusion. Behavioral testing Mechanical hyperalgesia Animals were habituated to the experimental procedure for 3 days before baseline testing. The room temperature and humidity remained stable for all experiments. All tests were performed during the light phase (8:00 am-16:00 pm). The experimenter who conducted the behavioral tests was blinded to all treatments. To determine mechanical nociceptive threshold, we measured the withdrawal response of the hind-paw to the electronic Von Frey filaments (Almemo
2450，Anesthesiometer IITC, Inc., Woodland Hills, CA) as previously described (Aguado et al., 2013). Rats were placed individually in a transparent plastic box (20 cm × 20 cm × 20 cm) with a wire mesh bottom (0.5 cm × 0.5cm) and allowed to habituate to the environment for 30 min before testing. Von Frey filaments were applied vertically to the plantar hind paw with sufficient pressure until a positive response was observed. A positive response was defined as a brisk withdrawal of paw. Each trial was repeated 3 times at 10 min interval and the mean threshold was obtained from the average value of the 3 trials. A maximal cut-off value of 60g was used to prevent tissue damage. Thermal hyperalgesia Thermal sensitivity was measured by using a Hargreaves radiant heat apparatus（SERIES 8, Model 390G, IITC Life Science, USA）. Rat were placed in clear plastic cage (20 cm × 15cm ×15 cm) with a glass floor and allowed to acclimatize for 30 min before testing. A radiant heat source was focused on the plantar surface of hind paw. The basal paw withdrawal latency was adjusted to 9 to 12 s and a cut-off time of 20s was used to avoid tissue damage. A positive response is defined as sudden paw withdrawal, flinching and/or paw licking. The trial was repeated 3 times at 10 min interval and the latency was recorded and averaged. Detection of mitochondrial superoxide using MitoSOX red MitoSOX Red (Molecular Probes, Eugene, OR, USA) was used to determine the levels of mitochondrial ROS according to the method previously described (Schwartz et al., 2008). Briefly, MitoSOX Red was dissolved in a 1:1 mixture of dimethylsulfoxide (DMSO) and saline to a final concentration of 33 µM. Approximately 24 h before remifentanil infusion, 20 µl of MitoSOX Red was intrathecal injected under isoflurane anesthesia. At the time of 6h, 1d, 3d after remifentanil treatment, rats were deeply anesthetized with isoflurane and perfused through the ascending aorta with saline, followed by 4% paraformaldehyde. After perfusion, the L4-5 segment of the spinal cord was removed and post-fixed in the same fixative for 2 h and then was placed in 30% sucrose for 48 h at 4 °C. Spinal cord sections (25 µm) were cut in a cryostat and examined under fluorescence microscope (Olympus BX51) with a rhodamine filter using a 100 × oil objective lens. The
intensity of MitoSOX positive cells in the spinal dorsal horn was analyzed using Image J (NIH, USA) analysis software. Immunohistochemistry Rats were deeply anesthetized with isoflurane and perfused through the ascending aorta with cold heparinized saline followed by 4% paraformaldehyde. The spinal cord of L4-L5 was removed and post-fixed in the same fixative for 2 h, then stored in 30% sucrose for 48 h at 4°C. Spinal cord was sectioned (25 µm) in a cryostat and stored in phosphate buffer saline (PBS, 0.01M) and processed for immunofluorescence. Sections were blocked with 5% donkey serum containing 0.3% Triton X-100 for 1 h at room temperature and then respectively incubated with the primary antibodies against CatS (1:400, SANT CRUZ), GFAP (astrocyte marker, 1:200, Millipore), OX-42 (microglia marker, 1:200, Millipore) or NeuN (neuronal marker, 1:200, Millipore) overnight at 4 °C. The sections were incubated with Cy3-conjugated secondary antibody (1:200, Jackson ImumunoResearch) or FITC-conjugated secondary antibody (1:200, Jackson ImumunoResearch) for 1 h. In some cases, spinal cord sections from the rats were used for double-staining with CatS and cellular markers. Also, the spinal cord sections from the rats injected with MitoSOX red were used for double-staining with cellular markers. The stained sections were examined under a fluorescence microscope (Olympus BX51, Japan) with Cy3 or FITC filters. The intensity of fluorescence was analyzed using Image J (NIH, USA) analysis software. Western blotting Rats were anesthetized with overdose of isoflurane at different time points following remifentanil infusion. L4–L5 segment of spinal cord was removed rapidly and stored in liquid nitrogen until used. Tissue samples were sonicated on ice in a lysis buffer containing a cocktail of protease inhibitors. The lysate was centrifuged at 13,000 rpm for 15 min at 4◦C and the supernatant was collected. Protein samples were separated by SDS-PAGE gel and transferred onto PVDF membrane. The membrane was blocked in TBS containing 5% nonfat milk and 0.1% Tween-20 for 2 h at room temperature and then incubated with primary antibodies for CatS (1:200, SANT
CRUZ), Iba1 (1:1000, Millipore), phosphorylated-NR1 (Ser896, 1:1000, Millipore) or NR1 (1:1000, Millipore) overnight at 4 °C. The membranes were washed with TBST buffer three times and incubated with HRP-conjugated secondary antibodies (anti-rabbit or anti-goat, 1:10000, Jackson ImmunoResearch) for 1 h at room temperature. ECL (Pierce, Rockford, IL) was used to detect the immune complex. To ensure equal protein loading, the membranes were stripped and incubated with primary antibodies for α-tubulin (1:10000, Sigma) or GAPDH (1:10000, Sigma). The relative expression of the protein bands was measured from scanned films using GS-700 Imaging Densitometer (GS-700, Bio-Rad Laboratories, Milan, Italy) and analyzed by using Image J (NIH, USA) analysis software. Statistical analyses All data experiments were expressed as mean±SEM. Repeated-measures ANOVA analysis of variance was performed to determine differences among groups at each time point. For analysis of immunohistochemistry and Western blot, differences between groups were compared by a one-way ANOVA followed by Fisher’s PLSD post hoc analysis. Significance was determined at a level of P < 0.05. Statistical analysis was performed with SPSS 21.0 (SPSS, USA).
RESULTS Continuous remifentanil infusion induces maturation of CatS in spinal microglia Consistent with previous report (Aguado et al., 2013, Ye et al., 2016), intravenous remifentanil infusion (4 µg·kg−1·min−1) for 2 hours induced significant reduction in withdrawal mechanical threshold (PWMT) (Fig. 1A) and paw withdrawal thermal latency (PWTL) (Fig. 1B). Mechanical allodynia and thermal hyperalgesia reached peak on day 1, maintained on day 3 and returned to the baseline on day 5, indicating establishment of remifentanil-induced hyperalgesia. Our and peers’ previous studies suggest that spinal microglia contribute to opioid-induced hyperalgesia (DeLeo et al., 2004, Cui et al., 2006b). In the present study, significant activation of spinal microglia was observed following remifentanil
infusion (Fig. 2A). Compared with vehicle group, activated microglia in remifentanil-treated rat displayed larger somal size and thicker processes. Growing evidence suggests that CatS in spinal microglia plays an important role in progression of neuropathic pain (Barclay et al., 2007, Clark et al., 2007). Then we set out to determine whether CatS expression is regulated following remifentanil infusion. In naive rats, CatS-immunopositive (ir) cells were identified in both grey and white matters of the spinal cord (Fig. 2B). Remifentanil treatment induced rapid but transient upregulation of CatS expression in the spinal cord, peaking within the first day and declined after day 3 (Fig. 2B, C) (P < 0.01). Furthermore, remifentanil treatment induced significant increase in the levels of both premature and mature forms of CatS (Fig. 2D, E) (P < 0.01). Double-staining showed that CatS-ir exclusively colocalized with OX-42 (a microglial marker), but not with NeuN (a neuronal marker) or GFAP (an astrocytic marker) (Fig. 2F). Inhibition of spinal CatS attenuates remifentanil-induced hyperalgesia To investigate the role of CatS in remifentanil-induced hyperalgesia, 50 nmol LHVS (an irreversible CatS inhibitor) was intrathecally delivered 30 min before remifentanil infusion. Intrathecal pretreatment with LHVS attenuated remifentanil-induced mechanical allodynia (Fig. 3 A) (P < 0.05) and thermal hyperalgesia (Fig. 3 B) (P < 0.05). LHVS alone had no effect on basal nociceptive threshold. In parallel with the inhibitory effect on remifentanil-induced hyperalgesia, increased OX-42 expression in the spinal cord was significantly reduced by LHVS pretreatment (Fig. 3C, D) (P < 0.01). Consistent with immunohistochemistry results, western blot analysis showed that LHVS inhibited the upregulation of Iba1 level induced by remifentanil (Fig. 3E, F) (P < 0.01). These data suggest that increased spinal CatS contributes to the development of mechanical allodynia and thermal hyperalgesia induced by remifentanil. Microglial CatS modulates phosphorylation of NMDA receptor following remifentanil treatment Fractalkine-mediated activation of microglia facilitates spinal synaptic transmission via modulation of NMDA receptor signaling (Clark et al., 2015) and activation of
NMDA receptor was found to be involved in remifentanil-induced hyperalgesia in our previous study (Ye et al., 2016). Therefore, upregulation of matured CatS in the present study leads us to determine whether microglial CatS contributes to remifentanil-induced hyperalgesia via NMDA signaling. Consistent with previous findings (Ye et al., 2016), enhanced phosphorylation of NMDA receptor NR1 subunit in the spinal cord was observed at 6 h and on day 1 following remifentanil treatment compared with vehicle group (Fig. 4A, B) (P < 0.01). However, intrathecal pretreatment with LHVS significantly prevented remifentanil-induced changes in NR1 phosphorylation (Fig. 4A, B) (P < 0.01), suggesting that contribution of CatS to remifentanil-induced hyperalgesia is associated with activation of NMDA receptor. Minocycline suppresses remifentanil-induced hyperalgesia and spinal CatS upregulation Compelling evidence has shown that minocycline, a specific microglia inhibitor, prevents development of morphine tolerance (Cui et al., 2008) and potentiates morphine analgesia (Mika et al., 2007). To evaluate the effect of minocycline on remifentanil-induced hyperalgesia, intrathecal delivery of minocycline (50 µg/ 10 µl) was performed 30 min before remifentanil infusion. As shown in Fig 5 A and B, remifentanil-induced mechanical allodynia and thermal hyperalgesia were attenuated by minocycline (P < 0.05). Minocycline alone had no significant effect on the basal mechanical and thermal withdrawal threshold. To define the mechanism by which minocycline attenuates remifentanil-induced hyperalgesia, we further examined the effect of minocycline on CatS expression in the spinal cord. Pretreatment with minocycline significantly reduced elevated expression of spinal CatS following remifentanil infusion (Fig. 5C, D) (P < 0.01). These data suggest that minocycline attenuates remifentanil-induced hyperalgesia by inhibiting CatS in the spinal microglia. Overproduction of ROS induces maturation of spinal CatS following remifentanil infusion ROS has been implied as an important mediator that induce activation of glial cells in several neuropathic diseases (Bezzi and Volterra, 2001, Watkins et al., 2001a) and
contributes to remifentanil-induced postoperative hyperalgesia (Shu et al., 2015). Then we examined whether spinal CatS was upregulated by overproduction of ROS following remifentanil infusion. Firstly we measured mitochondrial-derived superoxide production using superoxide-sensitive probe MitoSOX Red. As illustrated in Fig 6A and B, there was a significant increase in MitoSOX fluorescence intensity in the spinal dorsal horn following remifentanil infusion (P < 0.01), indicating that remifentanil induced excessive production of ROS. The level of ROS began to increase within 6 h, peaked on day 1 and declined on day 3 following remifentanil infusions. In addition, MitoSOX staining was exclusively colabeled with NeuN, but not with GFAP or OX-42 (Fig. 6C). Additionally, intravenous delivery of ROS scavenger PBN (20 and 100 mg/kg) administrated 30 min before remifentanil infusion significantly attenuated mechanical allodynia (Fig. 7A) (P < 0.05) and thermal hyperalgesia (Fig. 7B) (P < 0.05) in a dose-dependent manner. In parallel with the inhibitory effect on hyperalgesia, PBN (100 mg/kg) pretreatment significantly blocked increase in microglial CatS expression at 6 h and on day 1 following remifentanil treatment (Fig. 7C, D) (P < 0.01). In addition, we observed that PBN prevented increase in the level of mature form of CatS (Fig. 7E, F) (P < 0.01), while the increased level of premature form of CatS remains unchanged (Fig. 7E, G) (P >0.05), suggesting that neuronal ROS induced maturation of CatS in the spinal microglia.
DISCUSSION The principal finding of this study is that matured form of CatS is induced in the spinal
microglia
following
remifentanil
treatment
and
contributes
to
remifentanil-induced hyperalgesia via activation of NMDA signaling. Spinal administration of CatS inhibitor LHVS or microglia inhibitor minocycline attenuates remifentanil-induced hyperalgesia. Overproduction of ROS in spinal neurons promotes maturation of CatS from pre-forms to mature forms. The present study, for the first time, demonstrates a critical role of CatS in spinal microglia in the development of remifentanil-induced hyperalgesia.
Accumulating evidence from clinical and experimental studies has reported that continuous remifentanil infusion induced acute hyperalgesia in dose and time-dependent manners (Kim et al., 2015). Although multiple molecular factors involved in remifentanil-induced hyperalgesia have been addressed in decades of researches (Chu et al., 2008, Zhao and Joo, 2008, Gu et al., 2009, Lee et al., 2011, Wang et al., 2015), the underlying mechanisms still remain unclear. For the past two decades, the role of microglial cells in opioid-induced tolerance and hyperalgesia has gained much attention (Cui et al., 2006a, Cui et al., 2008, Mika, 2008, Hayashi et al., 2016, Roeckel et al., 2016). Upon activation, microglia produces proinflammatory cytokines such as TNF-α, IL-1β that contribute to central sensitization during chronic morphine therapy (Watkins et al., 2009). The present study showed rapid onset of hyperalgesia and significant activation of microglia in the spinal cord following remifentanil infusion. These results are consistent with recent finding that activation of microglia is induced during the process of remifentanil-induced postoperative hyperalgesia (Romero et al., 2013). Furthermore, inhibition of microglia activation by cannabinoid receptor type 2 (CB2) agonist attenuates remifentanil-induced hyperalgesia and suppresses expression of inflammatory cytokines (Sun et al., 2014). In the present study, we also found that microglia inhibitor minocycline suppressed mechanical allodynia and thermal hyperalgesia, providing further evidence that microgliosis plays a pivotal role in remifentanil-induced hyperalgesia. Cathepsin S (CatS) belongs to the family of lysosomal cysteine proteases and is associated with antigen presentation and extracellular protein turnover (Petanceska et al., 1996, Riese et al., 1998, Wilkinson et al., 2015). CatS is synthesized as an inactive preproenzyme and requires proteolytic removal of the N-terminal pro-peptide for activity (Clark et al., 2010). CatS has recently emerged as an important player in progression of pathogenesis of neuropathic pain. Upregulation of CatS in macrophages of dorsal root ganglia (Barclay et al., 2007) and in microglia of the spinal cord (Clark et al., 2007) has been detected after peripheral nerve injury. CatS inhibition or depletion attenuates neuropathic and inflammatory pain (Barclay et al., 2007, Clark et al., 2007, Clark and Malcangio, 2012). CatS exerts its pronociceptive
effects via cleavage of the neuronal chemokine fractalkine (Clark et al., 2007), while antagonizing fractalkine potentiates acute morphine analgesia and attenuates the development of hyperalgesia which occurs after chronic morphine treatment (Johnston et al., 2004). Here, we found that continuous remifentanil infusion leads to rapid, transient increase in the levels of both premature and mature forms of CatS in the activated spinal microglia, and the time course of CatS upregulation is comparable to the progression of hyperalgesia. Moreover, intrathecal administration of irreversible CatS inhibitor LHVS significantly attenuates remifentanil-induced hyperalgesia, as well as the activation of microglia. Therefore, our data support the hypothesis that CatS in spinal microglia contributes to enhanced pain sensitivity following remifentanil treatment. Several lines of evidence suggest that activation of NMDA receptor is involved in remifentanil-induced hyperalgesia (Gu et al., 2009, Yuan et al., 2013, Liu et al., 2014, Sun et al., 2014). Our previous study showed increased phosphorylation of NR1 NMDA receptor subunit following remifentanil treatment (Sun et al., 2014, Ye et al., 2016).
Interestingly,
pretreatment
with
CatS
inhibitor
also
blocked
remifentanil-induced phosphorylation of NR1 subunit, implying a possible mechanism by which microglial CatS leads to remifentanil-induced hyperalgesia. This hypothesis is well supported by recent report that fractalkine cleaved by CatS can induce the release of interleukin 1β from spinal microglia which can subsequently induce the phosphorylation of NMDA receptors (Clark et al., 2015). All these findings suggest that CatS released from spinal microglia is involved in modulation of NMDA receptors, thereby contributing to the development of remifentanil-induced hyperalgesia. It has been reported that the release of microglial CatS following activation of P2X7 receptor is downstream of p38 MAPK phosphorylation (Clark et al., 2010). In addition, inhibition of p38 activation by microglia inhibitor minocycline attenuates neuropathic pain (Clark et al., 2007) and morphine tolerance (Cui et al., 2008). Consistently, we observed that intrathecal delivery of minocycline significantly inhibits increase in the expression of CatS in microglia following remifentanil
treatment. Thus, it is possible that remifentanil might induce CatS upregulation via p38 MAPK pathway in spinal microglia. Evidence from macrophage and microglial cell lines demonstrate that CatS release can be induced following stimulation with lipopolysaccharide (LPS) (Clark et al., 2009) and inflammatory cytokines (Liuzzo et al., 1999). Recently, regulation of CatS expression and activity by reactive oxygen species (ROS) has been reported in several cell lines (Cheng et al., 2008, Seo et al., 2009, Tsai et al., 2014). As powerful pronociceptive mediators in oxidative stress and inflammation (Salvemini and Neumann, 2009), ROS contribute to the central sensitization associated with pain (Park et al., 2006, Gao et al., 2007, Lee et al., 2007, Kim et al., 2008, Salvemini et al., 2011) and are critical in the development of morphine tolerance/hyperalgesia (Muscoli et al., 2007, Doyle et al., 2009, Ndengele et al., 2009, Doyle et al., 2010, Little et al., 2013). Eliminating peroxynitrite with hydrogen-rich saline attenuates remifentanil-induced hyperalgesia (Shu et al., 2015). In accordance with previous study (Ye et al., 2016), we found that remifentanil treatment induced excessive production of ROS in spinal neurons，while pretreatment with non-selective ROS scavenger PBN markedly protects against remifentanil-induced hyperalgesia. In addition, our most finding is that PBN significantly prevents upregulation of matured form of CatS in microglia of the spinal cord, indicating that neuronal ROS plays a crucial role in promoting maturation of CatS. It is worth to note that remifentanil also induced an increase in the levels of premature forms of CatS which is not affected following removal of ROS by PBN. Therefore, other mechanisms responsible for such upregulation warrant further investigation.
CONCLUSIONS Our present study provides evidence that neuronal ROS promotes activity of microglial CatS which induces phosphorylation of NMDA receptor following remifentanil treatment. Despite the fact that ROS induces phosphorylation of NMDA receptor in rats treated by capsaicin (Gao et al., 2007) or remifentanil (Ye et al., 2016), the present findings suggest that ROS-CatS-NMDA signaling plays a critical role in
neuron-microglia positive feedback during development of remifentanil-induced hyperalgesia. Thus, microglial CatS in the spinal cord might serve as a novel therapeutic target for prevention of remifentanil-induced hyperalgesia.
COMPETING INTERESTS The authors have declared that no conflict of interest exists.
AUTHOR’S CONTRIBUTIONS L Ye and L Xiao carried out major work in the experiment. SY Yang performed the behavior testing. JJ Duan performed Western Blotting analysis. Y Chen discussed the results and commented on the manuscript. Y Cui and Y Chen supervised the experiments, analyzed the data and drafted the manuscript.
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LEGENDS Fig. 1. Remifentanil induced mechanical allodynia and thermal hyperalgesia. Significant reduction in mechanical withdrawal threshold (A) and thermal withdrawal latency (B) were seen following intravenous infusion of remifentanil (4 µg·kg−1·min−1 for 2 h). Data are presented as mean ± SEM (n= 8). *P < 0.05, compared with vehicle (Veh) group. Fig. 2. Remifentanil induces an increase in CatS expression in spinal microglia. (A) OX-42 immunostaining reveals activation of microglia as indicated by large cell bodies and thick process following remifentanil infusion. Scale bar = 50 µm. (B, C) Immunohistochemistry data show that CatS expression in the spinal cord is significantly induced by remifentanil treatment. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. (D, E) Western blot analysis reveals upregulation of premature and mature form of CatS in the spinal dorsal horn following remifentanil treatment. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. (F) Double-staining shows colocalization of CatS and OX-42 after remifentanil treatment. Scale bar = 50 µm. Fig. 3. Inhibition of CatS attenuates remifentanil-induced hyperalgesia and microglial activation. (A, B) Intrathecal administration of CatS inhibitor LHVS (50 nmol/10 ml) 30 min before remifentanil infusion reduces mechanical allodynia (A) and thermal hyperalgesia (B). Data are presented as mean ± SEM (n= 8), *P < 0.05, compared with remifentanil (R) group. (C, D) Immunohistochemistry data show that pretreatment with LHVS suppresses the activation of spinal microglia following remifentanil infusion. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. ##P < 0.01, compared with remifentanil group at the same time point. (E, F) Western blot shows upregulation of Iba1 levels in the spinal dorsal horn following remifentanil treatment is reduced by LHVS. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle. ##P < 0.01, compared with remifentanil group at the same time point. Fig. 4. Inhibition of CatS blocks phosphorylation of NMDA receptor NR1 subunit induced by remifentanil. (A) Western blot reveals that pretreatment with CatS
inhibitor reduces pNR1 but not total protein of NR1 in the spinal dorsal horn. (B) Quantification of relative density of pNR1 and NR1 among groups. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. ##P < 0.01, compared with remifentanil group at the same time point. Fig. 5. Intrathecal administration of microglia inhibitor minocycline attenuates remifentanil-induced hyperalgesia and CatS upregulation. (A, B) Spinal delivery of minocycline (50 mg/10 ml) 30 min before remifentanil infusion attenuates mechanical allodynia (A) and thermal hyperalgesia (B). Data are presented as mean ± SEM (n= 8), *P < 0.05, compared with remifentanil group. (C, D) Immunohistochemistry shows that minocycline suppresses upregulation of CatS in the spinal cord induced by remifentanil. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with remifentanil group at the same time point. Fig. 6. Remifentanil treatment induces overproduction of ROS in spinal neurons. (A) Production of ROS indicated by MitoSOX staining is induced in the spinal cord following remifentanil infusion. Scale bar: 20 µm. (B) Quantification of MitoSOX staining intensity in the spinal dorsal horn. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. (C) Double-staining shows exclusive colocalization of MitoSOX with NeuN. Scale bar: 20 µm. Fig. 7. Enhanced production of ROS by spinal neurons mediates upregulation of CatS following remifentanil infusion. (A, B) Systemic administration of PBN (20mg/kg or 100mg/kg) 30 min before remifentanil infusion significantly blocks the development of remifentanil-induced mechanical allodynia (A) and thermal hyperalgesia (B). Data are presented as mean ± SEM (n= 8), *P < 0.05 compared with remifentanil group. (C, D) ROS scavenger inhibits remifentanil-induced upregulation of CatS in the spinal dorsal horn. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01 compared with remifentanil group at the same time point. (E-G) Removal of ROS by PBN reduced upregulation of mature (E, F) but not premature (E, G) form of CatS induced by remifentanil. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with remifentanil group.
S0306-4522(16)30730-8 http://dx.doi.org/10.1016/j.neuroscience.2016.12.030 NSC 17511
To appear in:
Neuroscience
Received Date: Revised Date: Accepted Date:
2 August 2016 13 December 2016 18 December 2016
Please cite this article as: L. Ye, L. Xiao, SY. Yang, JJ. Duan, Y. Chen, Y. Cui, Y. Chen, Cathepsin S in the spinal microglia contributes to remifentanil-induced hyperalgesia in rats, Neuroscience (2016), doi: http://dx.doi.org/ 10.1016/j.neuroscience.2016.12.030
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Title: Cathepsin S in the spinal microglia contributes to remifentanil-induced hyperalgesia in rats Running Head: ROS/CatS signaling involved in remifentanil-induced hyperalgesia Authors: L. Yea,1, L. Xiaoa,1, SY. Yanga, JJ. Duanb, Y. Chenb, Y. Cuic, *, Y. Chena, ** Institutions: a Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510000, China b Neurobiology Research Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510000, China c Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510000, China Corresponding author: * Department of Physiology, Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan 2 Rd, Guangzhou, 510080, China. ** Department of Anesthesiology, First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan 2 Rd, Guangzhou, 510000, China Email: Email: [email protected] (Y Cui), [email protected] (Y Chen) 1
These authors contributed equally to this work
Category: Original article Founding sources: Science and Technology Project in Guangzhou (No. 2012B031800368; No. 2009B030801110), the Fundamental Research Funds for the Central Universities (NO.12ykpy03) Conflicts of interest: The authors have declared that no conflict of interest exists
Highlights 1. Remifentanil induces upregulation of matured cathepsin S (CatS) in spinal microglia 2. Contribution of CatS to remifentanil-induced hyperalgesia is linked to activation
of NMDA receptor 3. Minocycline inhibits CatS upregulation and hyperalgesia induced by remifentanil 4. Upregulation of matured CatS depends on ROS released from spinal neurons
ABSTRACT Cysteine protease Cathepsin S (CatS) expressed by spinal microglia has been shown to play a critical role in nerve injury and inflammation-induced chronic pain. However, whether microglial CatS contributes to remifentanil-induced acute hyperalgesia remains unstudied. In the present study, intravenous remifentanil infusion induced significant increase in the expression of premature and mature form of CatS in the activated microglia in the spinal cord. Spinal delivery of irreversible CatS inhibitor LHVS reduced hyperalgesia, attenuated activation of spinal microglia and blocked phosphorylation of NMDA receptor NR1 subunit induced by remifentanil. Furthermore, inhibition of microglia by minocycline effectively suppressed remifentanil-induced hyperalgesia, as well as CatS upregulation. In addition, remifentanil infusion also induced an increase in reactive oxygen species (ROS) levels in spinal neurons. Systemic administration of ROS scavenger PBN was sufficient to suppress remifentanil-induced painful hypersensitivity. Removal of ROS by PBN prevented upregulation of mature CatS in spinal microglia. However, increased protein level of premature form of CatS was not affected by PBN. Altogether, our findings demonstrate that neuronal ROS promote maturation of microglial CatS which facilitates activation of NMDA in the spinal dorsal horn. Therefore, such mechanism is involved in neuron-microglia positive feedback and contributes to remifentanil-induced hyperalgesia. Key words: remifentanil, hyperalgesia, Cathepsin S, microglia, minocycline, reactive oxygen species.
INTRODUCTION Remifentanil, a potent µ-opioid receptor agonist, is widely used for intraoperative anesthesia due to its rapid onset of action and clearance after withdrawal (Kim et al., 2014).
However,
hyperalgesia
may
develop
following
acute
remifentanil
administration and limits its potential clinical application (Joly et al., 2005, Bekhit, 2010). Although remifentanil-induced hyperalgesia has been reported in experimental studies and clinical observations in the past decade years (Hood et al., 2003), the mechanisms underlying such pathological process remain largely unknown. Growing evidence has suggested that spinal glial cells especially microglia modulate central sensitization (Watkins et al., 2001b, Obata et al., 2006) and thereby contributes to the development of opioid-induced tolerance (Cui et al., 2008) and hyperalgesia (Raghavendra et al., 2004, Ferrini and De Koninck, 2013). Recently, activation of spinal microglia and increased expression of cytokines are observed in the spinal cord following intraoperative remifentanil infusion (Romero et al., 2013, Sun et al., 2014). However, our understanding of the possible mechanism by which spinal microglia contributes to remifentanil-induced hyperalgesia is still limited. Cathepsin S (CatS) is a lysosomal cysteine protease expressed in macrophages, microglia and contributes to antigen presentation and adaptive immunity (Clark and Malcangio, 2012). CatS is initially synthesized as a pre-proenzyme and processed into its active mature form following removal of pro-peptide (Lecaille et al., 2002). Increased release of active CatS in spinal microglia has been detected in several neuropathic and inflammatory pain models (Clark et al., 2007, Clark et al., 2012, Nieto et al., 2016). Intriguingly, CatS has emerged as an important player in microglia-neuronal communication. It is found that selective activation of microglia by neuronal transmembrane fractalkine cleaved by CatS (Clark and Malcangio, 2012) contributes to increased pain sensitivity after morphine exposure (Johnston et al., 2004). Recently, fractalkine is found to induce the release of interleukin-1β (IL-1β) which modulates postsynaptic NMDA receptor signaling (Clark et al., 2015). Although activation of NMDA receptor in the spinal cord has been reported following remifentanil treatment (Zheng et al., 2012, Zhang et al., 2015, Ye et al., 2016),
whether neuron-microglia communication mediated by CatS-NMDA signaling in the spinal cord is involved in remifentanil-induced hyperalgesia has not been studied. The release of CatS from macrophage and microglia can be regulated by lipopolysaccharide (LPS) (Clark et al., 2009) and inflammatory cytokines (Liuzzo et al., 1999). The release of active CatS from microglia is also regulated by extracellular ATP via P2X7 receptor (Clark et al., 2010). Recently, regulation of CatS expression and activities by reactive oxygen species (ROS) has been reported in several cell lines (Cheng et al., 2008, Seo et al., 2009, Tsai et al., 2014). ROS are highly reactive metabolites of oxygen including superoxide, hydrogen peroxide and peroxynitrite (Dikalov and Harrison, 2014). Overproduction of ROS facilitates central sensitization and lead to hyperalgesia in several neuropathic pain models (Park et al., 2006, Gao et al., 2007, Lee et al., 2007, Kim et al., 2010). Of particular interest, it has been recently demonstrated that neuronal ROS is closely associated with the remifentanil-induced postoperative hyperalgesia (Zhang et al., 2014, Shu et al., 2015). Moreover, our recent report that ROS induces phosphorylation of NMDA receptor subunits following remifentanil treatment (Ye et al., 2016) further supports the role of ROS in remifentanil-induced
hyperalgesia.
Therefore,
whether
ROS
mediates
remifentanil-induced hyperalgesia via regulation of CatS in the spinal cord is worthy of investigation. In
the
present
study,
we
determined
whether
CatS
contributes
to
remifentanil-induced hyperalgesia via the mechanism associated with activation of NMDA signaling. Furthermore, we investigated regulation of CatS expression by neuronal ROS following remifentanil treatment in rats.
EXPERIMENTAL PROCEDURES Animals Male Sprague-Dawley rats (220-250g) were housed in a temperature-controlled room (22±1oC) with a 12-h light-dark cycle. All animals were acclimated for at least 1 week before any experimental procedures. All animal experimental procedures were approved by the Animal Care and Use Committee of Zhongshan school of Medicine,
and carried out in accordance with the guideline of National Institutes of Health on the animal care and the ethical guideline. Drug administration Remifentanil (Ren
Fu,
Co.,
Yichang,
China)
and
sevoflurane
(Maruishi
Pharmaceutical Co. Ltd, Japan) were supplied by the Department of Anesthesiology of the First Affiliated Hospital, Sun Yat-sen University (Guangzhou, China). Remifentanil was dissolved in saline (NaCl 0.9%) to a final concentration of 40 µg/ml. Following induction of anesthesia with sevoflurane (3.0% v/v) delivered via a nose mask, remifentanil were intravenous infused through tail vein over a period of 2 h (4 µg·kg−1·min−1) using an injection pump (Asena PK，UK). Control animals received the same volume of saline in identical conditions. ROS scavenger phenyl N-tert-butylnitrone (PBN; Sigma, St Louis, MO), CatS inhibitor LHVS (NeoMPS, San Diego, USA) or microglia inhibitor Minocycline （sigma，USA) was dissolved in saline. PBN (20 mg or 100 mg/kg) in total volume of 1 ml was injected intraperitoneally. LHVS (50 nmol/10 µl) or minocycline (50 µg/10 µl) was intrathecally injected as previous described (Mestre et al., 1994). In brief, the rats were anesthetized with sevoflurane and a 20 µl Hamilton microsyringe (GaoGe Co., Shanghai, China) was inserted between the L5 and L6 vertebrae. The entry of the needle into the subarachnoid space was confirmed when a sudden light tail flick occurred. Then LHVS or minocycline was injected into the subarachnoid space during a period of 30s, and the microsyringe was held for a further 10 s to prevent outflow of the agent. The inhibitor was given 30 min before remifentanil infusion. Behavioral testing Mechanical hyperalgesia Animals were habituated to the experimental procedure for 3 days before baseline testing. The room temperature and humidity remained stable for all experiments. All tests were performed during the light phase (8:00 am-16:00 pm). The experimenter who conducted the behavioral tests was blinded to all treatments. To determine mechanical nociceptive threshold, we measured the withdrawal response of the hind-paw to the electronic Von Frey filaments (Almemo
2450，Anesthesiometer IITC, Inc., Woodland Hills, CA) as previously described (Aguado et al., 2013). Rats were placed individually in a transparent plastic box (20 cm × 20 cm × 20 cm) with a wire mesh bottom (0.5 cm × 0.5cm) and allowed to habituate to the environment for 30 min before testing. Von Frey filaments were applied vertically to the plantar hind paw with sufficient pressure until a positive response was observed. A positive response was defined as a brisk withdrawal of paw. Each trial was repeated 3 times at 10 min interval and the mean threshold was obtained from the average value of the 3 trials. A maximal cut-off value of 60g was used to prevent tissue damage. Thermal hyperalgesia Thermal sensitivity was measured by using a Hargreaves radiant heat apparatus（SERIES 8, Model 390G, IITC Life Science, USA）. Rat were placed in clear plastic cage (20 cm × 15cm ×15 cm) with a glass floor and allowed to acclimatize for 30 min before testing. A radiant heat source was focused on the plantar surface of hind paw. The basal paw withdrawal latency was adjusted to 9 to 12 s and a cut-off time of 20s was used to avoid tissue damage. A positive response is defined as sudden paw withdrawal, flinching and/or paw licking. The trial was repeated 3 times at 10 min interval and the latency was recorded and averaged. Detection of mitochondrial superoxide using MitoSOX red MitoSOX Red (Molecular Probes, Eugene, OR, USA) was used to determine the levels of mitochondrial ROS according to the method previously described (Schwartz et al., 2008). Briefly, MitoSOX Red was dissolved in a 1:1 mixture of dimethylsulfoxide (DMSO) and saline to a final concentration of 33 µM. Approximately 24 h before remifentanil infusion, 20 µl of MitoSOX Red was intrathecal injected under isoflurane anesthesia. At the time of 6h, 1d, 3d after remifentanil treatment, rats were deeply anesthetized with isoflurane and perfused through the ascending aorta with saline, followed by 4% paraformaldehyde. After perfusion, the L4-5 segment of the spinal cord was removed and post-fixed in the same fixative for 2 h and then was placed in 30% sucrose for 48 h at 4 °C. Spinal cord sections (25 µm) were cut in a cryostat and examined under fluorescence microscope (Olympus BX51) with a rhodamine filter using a 100 × oil objective lens. The
intensity of MitoSOX positive cells in the spinal dorsal horn was analyzed using Image J (NIH, USA) analysis software. Immunohistochemistry Rats were deeply anesthetized with isoflurane and perfused through the ascending aorta with cold heparinized saline followed by 4% paraformaldehyde. The spinal cord of L4-L5 was removed and post-fixed in the same fixative for 2 h, then stored in 30% sucrose for 48 h at 4°C. Spinal cord was sectioned (25 µm) in a cryostat and stored in phosphate buffer saline (PBS, 0.01M) and processed for immunofluorescence. Sections were blocked with 5% donkey serum containing 0.3% Triton X-100 for 1 h at room temperature and then respectively incubated with the primary antibodies against CatS (1:400, SANT CRUZ), GFAP (astrocyte marker, 1:200, Millipore), OX-42 (microglia marker, 1:200, Millipore) or NeuN (neuronal marker, 1:200, Millipore) overnight at 4 °C. The sections were incubated with Cy3-conjugated secondary antibody (1:200, Jackson ImumunoResearch) or FITC-conjugated secondary antibody (1:200, Jackson ImumunoResearch) for 1 h. In some cases, spinal cord sections from the rats were used for double-staining with CatS and cellular markers. Also, the spinal cord sections from the rats injected with MitoSOX red were used for double-staining with cellular markers. The stained sections were examined under a fluorescence microscope (Olympus BX51, Japan) with Cy3 or FITC filters. The intensity of fluorescence was analyzed using Image J (NIH, USA) analysis software. Western blotting Rats were anesthetized with overdose of isoflurane at different time points following remifentanil infusion. L4–L5 segment of spinal cord was removed rapidly and stored in liquid nitrogen until used. Tissue samples were sonicated on ice in a lysis buffer containing a cocktail of protease inhibitors. The lysate was centrifuged at 13,000 rpm for 15 min at 4◦C and the supernatant was collected. Protein samples were separated by SDS-PAGE gel and transferred onto PVDF membrane. The membrane was blocked in TBS containing 5% nonfat milk and 0.1% Tween-20 for 2 h at room temperature and then incubated with primary antibodies for CatS (1:200, SANT
CRUZ), Iba1 (1:1000, Millipore), phosphorylated-NR1 (Ser896, 1:1000, Millipore) or NR1 (1:1000, Millipore) overnight at 4 °C. The membranes were washed with TBST buffer three times and incubated with HRP-conjugated secondary antibodies (anti-rabbit or anti-goat, 1:10000, Jackson ImmunoResearch) for 1 h at room temperature. ECL (Pierce, Rockford, IL) was used to detect the immune complex. To ensure equal protein loading, the membranes were stripped and incubated with primary antibodies for α-tubulin (1:10000, Sigma) or GAPDH (1:10000, Sigma). The relative expression of the protein bands was measured from scanned films using GS-700 Imaging Densitometer (GS-700, Bio-Rad Laboratories, Milan, Italy) and analyzed by using Image J (NIH, USA) analysis software. Statistical analyses All data experiments were expressed as mean±SEM. Repeated-measures ANOVA analysis of variance was performed to determine differences among groups at each time point. For analysis of immunohistochemistry and Western blot, differences between groups were compared by a one-way ANOVA followed by Fisher’s PLSD post hoc analysis. Significance was determined at a level of P < 0.05. Statistical analysis was performed with SPSS 21.0 (SPSS, USA).
RESULTS Continuous remifentanil infusion induces maturation of CatS in spinal microglia Consistent with previous report (Aguado et al., 2013, Ye et al., 2016), intravenous remifentanil infusion (4 µg·kg−1·min−1) for 2 hours induced significant reduction in withdrawal mechanical threshold (PWMT) (Fig. 1A) and paw withdrawal thermal latency (PWTL) (Fig. 1B). Mechanical allodynia and thermal hyperalgesia reached peak on day 1, maintained on day 3 and returned to the baseline on day 5, indicating establishment of remifentanil-induced hyperalgesia. Our and peers’ previous studies suggest that spinal microglia contribute to opioid-induced hyperalgesia (DeLeo et al., 2004, Cui et al., 2006b). In the present study, significant activation of spinal microglia was observed following remifentanil
infusion (Fig. 2A). Compared with vehicle group, activated microglia in remifentanil-treated rat displayed larger somal size and thicker processes. Growing evidence suggests that CatS in spinal microglia plays an important role in progression of neuropathic pain (Barclay et al., 2007, Clark et al., 2007). Then we set out to determine whether CatS expression is regulated following remifentanil infusion. In naive rats, CatS-immunopositive (ir) cells were identified in both grey and white matters of the spinal cord (Fig. 2B). Remifentanil treatment induced rapid but transient upregulation of CatS expression in the spinal cord, peaking within the first day and declined after day 3 (Fig. 2B, C) (P < 0.01). Furthermore, remifentanil treatment induced significant increase in the levels of both premature and mature forms of CatS (Fig. 2D, E) (P < 0.01). Double-staining showed that CatS-ir exclusively colocalized with OX-42 (a microglial marker), but not with NeuN (a neuronal marker) or GFAP (an astrocytic marker) (Fig. 2F). Inhibition of spinal CatS attenuates remifentanil-induced hyperalgesia To investigate the role of CatS in remifentanil-induced hyperalgesia, 50 nmol LHVS (an irreversible CatS inhibitor) was intrathecally delivered 30 min before remifentanil infusion. Intrathecal pretreatment with LHVS attenuated remifentanil-induced mechanical allodynia (Fig. 3 A) (P < 0.05) and thermal hyperalgesia (Fig. 3 B) (P < 0.05). LHVS alone had no effect on basal nociceptive threshold. In parallel with the inhibitory effect on remifentanil-induced hyperalgesia, increased OX-42 expression in the spinal cord was significantly reduced by LHVS pretreatment (Fig. 3C, D) (P < 0.01). Consistent with immunohistochemistry results, western blot analysis showed that LHVS inhibited the upregulation of Iba1 level induced by remifentanil (Fig. 3E, F) (P < 0.01). These data suggest that increased spinal CatS contributes to the development of mechanical allodynia and thermal hyperalgesia induced by remifentanil. Microglial CatS modulates phosphorylation of NMDA receptor following remifentanil treatment Fractalkine-mediated activation of microglia facilitates spinal synaptic transmission via modulation of NMDA receptor signaling (Clark et al., 2015) and activation of
NMDA receptor was found to be involved in remifentanil-induced hyperalgesia in our previous study (Ye et al., 2016). Therefore, upregulation of matured CatS in the present study leads us to determine whether microglial CatS contributes to remifentanil-induced hyperalgesia via NMDA signaling. Consistent with previous findings (Ye et al., 2016), enhanced phosphorylation of NMDA receptor NR1 subunit in the spinal cord was observed at 6 h and on day 1 following remifentanil treatment compared with vehicle group (Fig. 4A, B) (P < 0.01). However, intrathecal pretreatment with LHVS significantly prevented remifentanil-induced changes in NR1 phosphorylation (Fig. 4A, B) (P < 0.01), suggesting that contribution of CatS to remifentanil-induced hyperalgesia is associated with activation of NMDA receptor. Minocycline suppresses remifentanil-induced hyperalgesia and spinal CatS upregulation Compelling evidence has shown that minocycline, a specific microglia inhibitor, prevents development of morphine tolerance (Cui et al., 2008) and potentiates morphine analgesia (Mika et al., 2007). To evaluate the effect of minocycline on remifentanil-induced hyperalgesia, intrathecal delivery of minocycline (50 µg/ 10 µl) was performed 30 min before remifentanil infusion. As shown in Fig 5 A and B, remifentanil-induced mechanical allodynia and thermal hyperalgesia were attenuated by minocycline (P < 0.05). Minocycline alone had no significant effect on the basal mechanical and thermal withdrawal threshold. To define the mechanism by which minocycline attenuates remifentanil-induced hyperalgesia, we further examined the effect of minocycline on CatS expression in the spinal cord. Pretreatment with minocycline significantly reduced elevated expression of spinal CatS following remifentanil infusion (Fig. 5C, D) (P < 0.01). These data suggest that minocycline attenuates remifentanil-induced hyperalgesia by inhibiting CatS in the spinal microglia. Overproduction of ROS induces maturation of spinal CatS following remifentanil infusion ROS has been implied as an important mediator that induce activation of glial cells in several neuropathic diseases (Bezzi and Volterra, 2001, Watkins et al., 2001a) and
contributes to remifentanil-induced postoperative hyperalgesia (Shu et al., 2015). Then we examined whether spinal CatS was upregulated by overproduction of ROS following remifentanil infusion. Firstly we measured mitochondrial-derived superoxide production using superoxide-sensitive probe MitoSOX Red. As illustrated in Fig 6A and B, there was a significant increase in MitoSOX fluorescence intensity in the spinal dorsal horn following remifentanil infusion (P < 0.01), indicating that remifentanil induced excessive production of ROS. The level of ROS began to increase within 6 h, peaked on day 1 and declined on day 3 following remifentanil infusions. In addition, MitoSOX staining was exclusively colabeled with NeuN, but not with GFAP or OX-42 (Fig. 6C). Additionally, intravenous delivery of ROS scavenger PBN (20 and 100 mg/kg) administrated 30 min before remifentanil infusion significantly attenuated mechanical allodynia (Fig. 7A) (P < 0.05) and thermal hyperalgesia (Fig. 7B) (P < 0.05) in a dose-dependent manner. In parallel with the inhibitory effect on hyperalgesia, PBN (100 mg/kg) pretreatment significantly blocked increase in microglial CatS expression at 6 h and on day 1 following remifentanil treatment (Fig. 7C, D) (P < 0.01). In addition, we observed that PBN prevented increase in the level of mature form of CatS (Fig. 7E, F) (P < 0.01), while the increased level of premature form of CatS remains unchanged (Fig. 7E, G) (P >0.05), suggesting that neuronal ROS induced maturation of CatS in the spinal microglia.
DISCUSSION The principal finding of this study is that matured form of CatS is induced in the spinal
microglia
following
remifentanil
treatment
and
contributes
to
remifentanil-induced hyperalgesia via activation of NMDA signaling. Spinal administration of CatS inhibitor LHVS or microglia inhibitor minocycline attenuates remifentanil-induced hyperalgesia. Overproduction of ROS in spinal neurons promotes maturation of CatS from pre-forms to mature forms. The present study, for the first time, demonstrates a critical role of CatS in spinal microglia in the development of remifentanil-induced hyperalgesia.
Accumulating evidence from clinical and experimental studies has reported that continuous remifentanil infusion induced acute hyperalgesia in dose and time-dependent manners (Kim et al., 2015). Although multiple molecular factors involved in remifentanil-induced hyperalgesia have been addressed in decades of researches (Chu et al., 2008, Zhao and Joo, 2008, Gu et al., 2009, Lee et al., 2011, Wang et al., 2015), the underlying mechanisms still remain unclear. For the past two decades, the role of microglial cells in opioid-induced tolerance and hyperalgesia has gained much attention (Cui et al., 2006a, Cui et al., 2008, Mika, 2008, Hayashi et al., 2016, Roeckel et al., 2016). Upon activation, microglia produces proinflammatory cytokines such as TNF-α, IL-1β that contribute to central sensitization during chronic morphine therapy (Watkins et al., 2009). The present study showed rapid onset of hyperalgesia and significant activation of microglia in the spinal cord following remifentanil infusion. These results are consistent with recent finding that activation of microglia is induced during the process of remifentanil-induced postoperative hyperalgesia (Romero et al., 2013). Furthermore, inhibition of microglia activation by cannabinoid receptor type 2 (CB2) agonist attenuates remifentanil-induced hyperalgesia and suppresses expression of inflammatory cytokines (Sun et al., 2014). In the present study, we also found that microglia inhibitor minocycline suppressed mechanical allodynia and thermal hyperalgesia, providing further evidence that microgliosis plays a pivotal role in remifentanil-induced hyperalgesia. Cathepsin S (CatS) belongs to the family of lysosomal cysteine proteases and is associated with antigen presentation and extracellular protein turnover (Petanceska et al., 1996, Riese et al., 1998, Wilkinson et al., 2015). CatS is synthesized as an inactive preproenzyme and requires proteolytic removal of the N-terminal pro-peptide for activity (Clark et al., 2010). CatS has recently emerged as an important player in progression of pathogenesis of neuropathic pain. Upregulation of CatS in macrophages of dorsal root ganglia (Barclay et al., 2007) and in microglia of the spinal cord (Clark et al., 2007) has been detected after peripheral nerve injury. CatS inhibition or depletion attenuates neuropathic and inflammatory pain (Barclay et al., 2007, Clark et al., 2007, Clark and Malcangio, 2012). CatS exerts its pronociceptive
effects via cleavage of the neuronal chemokine fractalkine (Clark et al., 2007), while antagonizing fractalkine potentiates acute morphine analgesia and attenuates the development of hyperalgesia which occurs after chronic morphine treatment (Johnston et al., 2004). Here, we found that continuous remifentanil infusion leads to rapid, transient increase in the levels of both premature and mature forms of CatS in the activated spinal microglia, and the time course of CatS upregulation is comparable to the progression of hyperalgesia. Moreover, intrathecal administration of irreversible CatS inhibitor LHVS significantly attenuates remifentanil-induced hyperalgesia, as well as the activation of microglia. Therefore, our data support the hypothesis that CatS in spinal microglia contributes to enhanced pain sensitivity following remifentanil treatment. Several lines of evidence suggest that activation of NMDA receptor is involved in remifentanil-induced hyperalgesia (Gu et al., 2009, Yuan et al., 2013, Liu et al., 2014, Sun et al., 2014). Our previous study showed increased phosphorylation of NR1 NMDA receptor subunit following remifentanil treatment (Sun et al., 2014, Ye et al., 2016).
Interestingly,
pretreatment
with
CatS
inhibitor
also
blocked
remifentanil-induced phosphorylation of NR1 subunit, implying a possible mechanism by which microglial CatS leads to remifentanil-induced hyperalgesia. This hypothesis is well supported by recent report that fractalkine cleaved by CatS can induce the release of interleukin 1β from spinal microglia which can subsequently induce the phosphorylation of NMDA receptors (Clark et al., 2015). All these findings suggest that CatS released from spinal microglia is involved in modulation of NMDA receptors, thereby contributing to the development of remifentanil-induced hyperalgesia. It has been reported that the release of microglial CatS following activation of P2X7 receptor is downstream of p38 MAPK phosphorylation (Clark et al., 2010). In addition, inhibition of p38 activation by microglia inhibitor minocycline attenuates neuropathic pain (Clark et al., 2007) and morphine tolerance (Cui et al., 2008). Consistently, we observed that intrathecal delivery of minocycline significantly inhibits increase in the expression of CatS in microglia following remifentanil
treatment. Thus, it is possible that remifentanil might induce CatS upregulation via p38 MAPK pathway in spinal microglia. Evidence from macrophage and microglial cell lines demonstrate that CatS release can be induced following stimulation with lipopolysaccharide (LPS) (Clark et al., 2009) and inflammatory cytokines (Liuzzo et al., 1999). Recently, regulation of CatS expression and activity by reactive oxygen species (ROS) has been reported in several cell lines (Cheng et al., 2008, Seo et al., 2009, Tsai et al., 2014). As powerful pronociceptive mediators in oxidative stress and inflammation (Salvemini and Neumann, 2009), ROS contribute to the central sensitization associated with pain (Park et al., 2006, Gao et al., 2007, Lee et al., 2007, Kim et al., 2008, Salvemini et al., 2011) and are critical in the development of morphine tolerance/hyperalgesia (Muscoli et al., 2007, Doyle et al., 2009, Ndengele et al., 2009, Doyle et al., 2010, Little et al., 2013). Eliminating peroxynitrite with hydrogen-rich saline attenuates remifentanil-induced hyperalgesia (Shu et al., 2015). In accordance with previous study (Ye et al., 2016), we found that remifentanil treatment induced excessive production of ROS in spinal neurons，while pretreatment with non-selective ROS scavenger PBN markedly protects against remifentanil-induced hyperalgesia. In addition, our most finding is that PBN significantly prevents upregulation of matured form of CatS in microglia of the spinal cord, indicating that neuronal ROS plays a crucial role in promoting maturation of CatS. It is worth to note that remifentanil also induced an increase in the levels of premature forms of CatS which is not affected following removal of ROS by PBN. Therefore, other mechanisms responsible for such upregulation warrant further investigation.
CONCLUSIONS Our present study provides evidence that neuronal ROS promotes activity of microglial CatS which induces phosphorylation of NMDA receptor following remifentanil treatment. Despite the fact that ROS induces phosphorylation of NMDA receptor in rats treated by capsaicin (Gao et al., 2007) or remifentanil (Ye et al., 2016), the present findings suggest that ROS-CatS-NMDA signaling plays a critical role in
neuron-microglia positive feedback during development of remifentanil-induced hyperalgesia. Thus, microglial CatS in the spinal cord might serve as a novel therapeutic target for prevention of remifentanil-induced hyperalgesia.
COMPETING INTERESTS The authors have declared that no conflict of interest exists.
AUTHOR’S CONTRIBUTIONS L Ye and L Xiao carried out major work in the experiment. SY Yang performed the behavior testing. JJ Duan performed Western Blotting analysis. Y Chen discussed the results and commented on the manuscript. Y Cui and Y Chen supervised the experiments, analyzed the data and drafted the manuscript.
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LEGENDS Fig. 1. Remifentanil induced mechanical allodynia and thermal hyperalgesia. Significant reduction in mechanical withdrawal threshold (A) and thermal withdrawal latency (B) were seen following intravenous infusion of remifentanil (4 µg·kg−1·min−1 for 2 h). Data are presented as mean ± SEM (n= 8). *P < 0.05, compared with vehicle (Veh) group. Fig. 2. Remifentanil induces an increase in CatS expression in spinal microglia. (A) OX-42 immunostaining reveals activation of microglia as indicated by large cell bodies and thick process following remifentanil infusion. Scale bar = 50 µm. (B, C) Immunohistochemistry data show that CatS expression in the spinal cord is significantly induced by remifentanil treatment. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. (D, E) Western blot analysis reveals upregulation of premature and mature form of CatS in the spinal dorsal horn following remifentanil treatment. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. (F) Double-staining shows colocalization of CatS and OX-42 after remifentanil treatment. Scale bar = 50 µm. Fig. 3. Inhibition of CatS attenuates remifentanil-induced hyperalgesia and microglial activation. (A, B) Intrathecal administration of CatS inhibitor LHVS (50 nmol/10 ml) 30 min before remifentanil infusion reduces mechanical allodynia (A) and thermal hyperalgesia (B). Data are presented as mean ± SEM (n= 8), *P < 0.05, compared with remifentanil (R) group. (C, D) Immunohistochemistry data show that pretreatment with LHVS suppresses the activation of spinal microglia following remifentanil infusion. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. ##P < 0.01, compared with remifentanil group at the same time point. (E, F) Western blot shows upregulation of Iba1 levels in the spinal dorsal horn following remifentanil treatment is reduced by LHVS. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle. ##P < 0.01, compared with remifentanil group at the same time point. Fig. 4. Inhibition of CatS blocks phosphorylation of NMDA receptor NR1 subunit induced by remifentanil. (A) Western blot reveals that pretreatment with CatS
inhibitor reduces pNR1 but not total protein of NR1 in the spinal dorsal horn. (B) Quantification of relative density of pNR1 and NR1 among groups. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. ##P < 0.01, compared with remifentanil group at the same time point. Fig. 5. Intrathecal administration of microglia inhibitor minocycline attenuates remifentanil-induced hyperalgesia and CatS upregulation. (A, B) Spinal delivery of minocycline (50 mg/10 ml) 30 min before remifentanil infusion attenuates mechanical allodynia (A) and thermal hyperalgesia (B). Data are presented as mean ± SEM (n= 8), *P < 0.05, compared with remifentanil group. (C, D) Immunohistochemistry shows that minocycline suppresses upregulation of CatS in the spinal cord induced by remifentanil. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with remifentanil group at the same time point. Fig. 6. Remifentanil treatment induces overproduction of ROS in spinal neurons. (A) Production of ROS indicated by MitoSOX staining is induced in the spinal cord following remifentanil infusion. Scale bar: 20 µm. (B) Quantification of MitoSOX staining intensity in the spinal dorsal horn. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with vehicle group. (C) Double-staining shows exclusive colocalization of MitoSOX with NeuN. Scale bar: 20 µm. Fig. 7. Enhanced production of ROS by spinal neurons mediates upregulation of CatS following remifentanil infusion. (A, B) Systemic administration of PBN (20mg/kg or 100mg/kg) 30 min before remifentanil infusion significantly blocks the development of remifentanil-induced mechanical allodynia (A) and thermal hyperalgesia (B). Data are presented as mean ± SEM (n= 8), *P < 0.05 compared with remifentanil group. (C, D) ROS scavenger inhibits remifentanil-induced upregulation of CatS in the spinal dorsal horn. Scale bar = 200 µm. Data are presented as mean ± SEM (n= 4), **P < 0.01 compared with remifentanil group at the same time point. (E-G) Removal of ROS by PBN reduced upregulation of mature (E, F) but not premature (E, G) form of CatS induced by remifentanil. Data are presented as mean ± SEM (n= 4), **P < 0.01, compared with remifentanil group.