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mTOR pathway is involved in ADP-evoked astrocyte activation and ATP release in the spinal dorsal horn in a rat neuropathic pain model
Neuroscience 275 (2014) 395–403
mTOR PATHWAY IS INVOLVED IN ADP-EVOKED ASTROCYTE ACTIVATION AND ATP RELEASE IN THE SPINAL DORSAL HORN IN A RAT NEUROPATHIC PAIN MODEL J. CUI, a W. HE, b B. YI, a H. ZHAO, c K. LU, a* H. RUAN b* AND D. MA c
Key words: adenosine triphosphate, adenosine diphosphate, astrocyte, mammalian target of rapamycin, neuropathic pain.
a Department of Anaesthesiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China b Department of Neurobiology, Chongqing Key Laboratory of Neurobiology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China
INTRODUCTION Neuropathic pain often develops when nerves are damaged through diabetes, surgery, infection and physical injury, and does not resolve upon the healing of the overt damaged tissue (Costigan et al., 2009). The current clinical management of neuropathic pain is not optimal and in fact, patients are relatively unresponsive to most available treatments, such as anticonvulsive, antidepressant and even opioids (Hearn et al., 2014). Mounting evidence has indicated that neuropathic pain is induced by the aberrant excitability of dorsal horn neurons evoked by peripheral sensory inputs (Costigan et al., 2009). Apart from molecular and cellular alterations in primary sensory neurons and in the spinal dorsal horn after nerve injury, recent studies have identified that spinal astrocyte activation, termed the reactive astrogliosis (Karimi-Abdolrezaee and Billakanti, 2012), enhances the production of various mediators, including proinflammatory cytokines (Brambilla et al., 2005) and brain-derived neurotrophic factor (Zhang et al., 2011), which promotes the hyperexcitability of dorsal horn neurons (Gwak and Hulsebosch, 2009). Reactive astrogliosis has been shown as a reflection of the severity of peripheral nerve injury and is characterized by cellular hypertrophy, increased glial fibrillary acidic protein (GFAP) expression and cellular proliferation (Cirillo et al., 2012). Molecular mechanisms that control the initiation of the development of the reactive astrogliosis are, nonetheless, poorly understood. Recently, studies have demonstrated that the extracellular ATP could act as a crucial neuromodulator on glial cell activation in the central nervous system and plays an important role in the neuropathic pain developed from peripheral nerve injury (Jakobsson, 2010). The mechanism of ATP release from certain cells and the effects of its degradation (ADP and AMP) on the induction of glial cell activation have recently attracted more attention in pain research (Burnstock, 2012). P2X ion channels and P2Y G protein-coupled receptors have been identified on neural cells, which could be activated by extracellular ATP (Burnstock et al., 2011). ADP is the primary enzymatic breakdown product in extracellular space and only contributes to the activation of P2Y receptors (Malin and
c
Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea & Westminster Hospital, London, UK
Abstract—Background: ATP/ADP-evoked spinal astrocyte activation plays a vital role in the development of neuropathic pain. We aim to investigate the role of mammalian target of rapamycin (mTOR) pathway on the spinal astrocyte activation in the neuropathic pain development in rats. Methods: Sprague Dawley (SD) rats were subjected to chronic constriction of the sciatic nerve (CCI). Rapamycin or ADP was intrathecally injected daily to explore their effects on spinal astrocyte activation and pain development. Expression of glial fibrillary acidic protein (GFAP) and mTOR in the spinal dorsal horn was assessed by immunohistochemistry. Von Frey hairs and Hargreaves paw withdrawal test were conducted to evaluate mechanical allodynia and thermal sensitivity, respectively. Firefly luciferase ATP assay was used to assess the change of ATP level in cerebrospinal fluid (CSF) and medium of cultured astrocytes. Results: GFAP expression was enhanced in the ipsilateral spinal dorsal horn from day 3 after surgery. GFAP and mTOR expression in the rat spinal dorsal horn on post-surgical day 14 was enhanced by daily intrathecal injection of ADP, which was inhibited by rapamycin. Rapamycin decreased lower mechanical pain threshold and the thermal withdrawal latency. Intrathecal injection of ADP enhanced the ATP release, which was partially inhibited by rapamycin. Study of cultured astrocytes indicated that ATP could be released from astrocytes. Conclusion: Our data demonstrated that ADP enhanced neuropathic pain in CCI rats, which was inhibited by rapamycin. This study indicates that targeting mTOR pathway could serve as a novel therapeutic strategy in neuropathic pain management. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding authors. Tel/fax: +86-23-68754197 (K. Lu), +8623-68753672 (H. Ruan). E-mail addresses: [email protected] (K. Lu), [email protected] (H. Ruan). Abbreviations: AMPK, AMP-activated protein kinase; CCI, chronic constriction injury model; CSF, cerebrospinal fluid; GFAP, glial fibrillary acidic protein; mTOR, mammalian target of rapamycin; SD, Sprague Dawley; vFh, Von Frey hairs. http://dx.doi.org/10.1016/j.neuroscience.2014.06.030 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 395
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Molliver, 2010). However, it remains unclear whether ADP could activate glial cells and induce neuropathic pain after peripheral nerve injury. Mammalian target of rapamycin (mTOR) pathway has been found to play a vital role in controlling local protein synthesis in neuronal axons and dendrites, as well as in modulating memory processes and plasticity (Takei et al., 2004). Rapamycin, an inhibitor of mTOR, has been reported to inhibit the activation of glial cells in the spinal cord and brain (Norsted Gregory et al., 2013; Oubrahim et al., 2013). Furthermore, it has been shown that neuropathic pain in mice was reduced through intraperitoneal injection of rapamycin (Obara et al., 2011). In addition, mTOR pathway plays an important role in transmitting the signal from G-coupled protein to mitochondria, which regulates many cellular activities such as cell proliferation and cytokine release (Villa-Cuesta et al., 2014). Recently, mTOR has become a therapeutic target in the activation of neural cells (Morgan-Warren et al., 2013). Given the pivotal role of astrocytes in promoting and sustaining chronic neuropathic pain, targeting astrocytes could reveal novel therapies for the clinical management. We have previously demonstrated that ADP-evoked extracellular ATP accumulation around astrocytes is mediated by P2Y receptors and AMPactivated protein kinase (AMPK) (Cui et al., 2011). AMPK and mTOR signaling pathways are associated with the ATP synthesis and consumption in cytoplasm and targeting both pathways has been shown to be beneficial for the treatment with pain (Melemedjian et al., 2011; Obara et al., 2011). In the present study, we aim to explore the role of the mTOR signaling pathway in the ATP release from the astrocytes through using the rat chronic constriction injury model (CCI), a well-established model of neuropathic pain (Bennett et al., 2003). Furthermore, the painrelieving effect of rapamycin on ATP/ADP-mediated neuropathic pain was also evaluated.
EXPERIMENTAL PROCEDURE Animals and surgery Adult female Sprague Dawley (SD) rats (200–250 g) were used for establishing neuropathic pain model (Tall et al., 2001) and post-natal SD rats (1–3 days old) were used to prepare astrocyte culture of the spinal cord. The rats were provided by the Center of Laboratory Animals of Third Military Medical University, Chongqing, China. All experiment protocols were approved by the Ethics Committee of Third Military Medical University. Efforts have been made to reduce animal suffering and minimize animal number used. Animals were housed in plastic cages with free access to food and water under a 12-h light/dark cycle. Intrathecal catheters were implanted as previously described (Yang et al., 2012). Briefly, adult female SD rats were anesthetized with 3.5% chloral hydrate (10 ml/kg, i.p.). The L4–L5 vertebra of the rat was opened on the dorsal side, and then the L5 spinouts process was removed. A polyethylene catheter (PE-10, BD, USA) was implanted to a depth of about 1.0 cm from the incision between the gaps of L4 and L5 vertebra and the surgical wound was sutured. The other end of the catheter was
exited at the back neck of the rat via a subcutaneous tunnel and was secured in situ. The correct intrathecal catheter positioning was confirmed with the paralysis of the bilateral hind limbs after 2% lidocaine (10 ll) injection through the catheter. Those with success of catheter positioning were allowed for 3–5-day recovery before the right sciatic nerve ligation surgery (Bennett and Xie, 1988). Briefly, the right common sciatic nerve branches (close to the trifurcation into the sural, peroneal and tibial nerve) were exposed and tied with 4–0 cotton sutures under chloral hydrate (3.5%) anesthesia. After intrathecal catheter was implanted successfully, rats without any other surgeries served as the naı¨ ve control. Rats with right sciatic nerve exposure without ligation served as the sham controls. Animals were used for experiments for up to 21 days after CCI surgery. Drug administration Rats were administered intrathecally with 10-ll rapamycin (100 nM, Merck Millipore, USA) or saline (10 ll as vehicle control), and 15 min later, 10 ll ADP (1 mM, Sigma, USA) was administered through the same route. The drugs were used once a day immediately after nerve injury until post-surgical day 14. Mechanical and thermal sensitivity measurement Von Frey hairs test (vFh) with bending forces at a range of 0.3–20.3 g was used to assess mechanical allodynia (He et al., 2012). The test began with the 4.10-g vFh (the middle of the filament series). The ventral surface of each hind paw was subjected to the filament for 4–6 s. If the animal lifted the hind paw quickly, it was recorded as a positive paw withdrawal response. The up-down method was used to determine the 50% paw-withdrawal mechanical threshold (PWMT). Hargreaves paw withdrawal was used to assess thermal sensitivity. It measured the withdrawal latency from a radiant heat source applied on the proximal half of the plantar surface of each hind paw (Han et al., 2011). The paw-withdrawal thermal latency (PWTL) was recorded as the threshold of thermal sensitivity. Each hind paw was tested five times in every 5 min. Immunohistochemistry Under terminated anesthesia (chloral hydrate 350 mg/kg, i.p.), rats were perfused intracardially with 4% paraformaldehyde. Spinal cords were quickly removed from rats and fixed overnight. After dehydration and cryoprotection for 48 h in 30% sucrose at 4 °C, the L4–6 segments of the frozen spinal cords were sectioned into 16-lm slices. After inhibition of endogenous peroxidase with 3% H2O2 for 15 min (this process will be omitted for immune-fluorescence staining), the slices were incubated with 10% goat serum for 1 h and then incubated with primary rabbit anti-rat mTOR (1:250, Millipore, USA) antibody and mouse anti-rat GFAP antibody (1:300, Sigma, USA) overnight at 4 °C. For ex vivo DAB staining, the antibody was visualized with a nickel-intensified DAB chromogen and the images were
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obtained with a microscope (Leica, Wetzlar, Germany). For the immune-fluorescence, the section was incubated with Cy3-conjugated goat anti-rabbit antibody (1:300, Jackson Immuno-Research, West Grove, PA, USA) and FITC-conjugated goat anti-mouse antibody (1:300, Jackson Immuno-Research) for 1 h. The nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, 1:1000, Sigma, USA) and examined with a laser scanning confocal microscope (Olympus FLV1000, Germany). Five randomly selected spinal sections were chosen for each animal, and the mean intensity of each section was analyzed with Image J software (NIH, US National Institutes of Health, Bethesda, MD). Data were expressed as the percentage relative to that of the naı¨ ve controls. Spinal astrocyte culture The primary culture of dorsal spinal cord astrocytes was prepared (Zeng et al., 2008). Briefly, postnatal SD rats (1–3 days old) were killed by decapitation and spinal cords were removed quickly. Meninges were removed under dissecting microscope and L1–L6 sections were harvested. The spinal cords were sectioned and then dissociated with 0.25% trypsin (Sigma, USA) for 15 min at 37 °C. The cells were cultured in the DMEM-F12 medium supplemented with 10% fetal calf serum and 50U/ml penicillin, 5 lg/ml streptomycin (Sigma–Aldrich, USA) for 10 days and then centrifuged at 450 rpm for 12 h to remove microglia and neuron. The quality of astrocyte culture was confirmed through using GFAP staining and astrocyte purity over 95% was used. ATP level assessment The ATP levels in rat cerebrospinal fluid (CSF) and cell culture medium were assessed through the firefly luciferase ATP assay kit (Beyotime, China) as described previously (Cui et al., 2011). Briefly, astrocytes cultured in 6-well plates were treated with 100 lM GDPbetaS (antagonist of P2Y receptors) or 10 nM rapamycin for 15 min and then exposed to 100 lM ADP for additional 24 h. Culture medium was collected from the astrocyte culture medium and kept at 80 °C before detection. Rat CSF was collected through a 22-G needle punctured into the L4–L5 spinal space of rats close to intrathecal catheter. 150-ll CSF or 500-ll culture medium was placed in 1.5-ml Eppendorf tubes and then centrifuged at 12,000g for 5 min at 4 °C. After centrifugation, 100 ll of the supernatant was mixed with 100 ll of ATP detection solution at its working dilution in a 1.5-ml Eppendorf tube. Luminance (RLU) was measured using a GloMax 20/20 luminometer (Promega, USA) and the concentration of ATP was measured. Statistical analysis All data were presented as mean ± SEM. Data were collected from at least four independent experiments (n P 4) and were analyzed statistically with a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc tests if multiple
comparisons were necessary (SPSS16.0, SPSS Inc., Chicago, IL, USA). A P-value of 0.05 was considered as statistically significant.
RESULTS Astrocytes were activated in the spinal dorsal horn after CCI The GFAP expression, a typical marker for astrocyte activation, was assessed in the ipsilateral spinal dorsal horn in the rats after CCI through immunohistochemistry. The expression of GFAP was enhanced gradually in the ipsilateral spinal dorsal horn from day 3 after the partial sciatic nerve ligation of rats when compared with that of controls (Fig. 1A). The intensity of GFAP staining was increased to the highest level 14 days after CCI surgery (Fig. 1B). ADP further enhanced astrocyte activation which was attenuated by rapamycin administration To investigate the effects of ADP and rapamycin on the activation of spinal astrocytes in CCI rats, rapamycin and ADP were injected into the spinal space daily through the intrathecal tube embedded in advance. The increase in GFAP expression that was observed in the rat spinal dorsal horn on the day 14 after the sciatic nerve ligation operation was enhanced by injection of 10 nmol ADP (P < 0.01, 472.73 ± 36.39% vs 312.65 ± 35.01% in CCI rats without ADP treatment). This high expression of GFAP induced by ADP was inhibited by pre-treatment with 0.01-nmol rapamycin (P < 0.001, 198.36 ± 25.41% vs 472.73 ± 36.39% in the ADP-treated CCI rats) (Fig. 2). Enhanced expression of mTOR was attenuated by rapamycin on the spinal astrocytes of CCI rats Expression of mTOR on the spinal astrocytes of CCI rats was assessed with a dual-labeling immune-fluorescence assay. mTOR and GFAP fluorescence staining were found to be co-localized (Fig. 3A, B), indicating enhanced expression of mTOR in activated astrocytes. The expression of mTOR was significantly increased on day 14 after CCI operation (137.61 ± 3.51% relative to control), which was enhanced by intrathecal injection with ADP (P < 0.01, 178.51 ± 18.05% vs 137.61 ± 3.51% in CCI rats), furthermore, this increase in expression could be suppressed by intrathecal injection of rapamycin (P < 0.001, 128.56 ± 8.67% vs 178.51 ± 18.05% in CCI rats treated with ADP) (Fig. 3C). Rapamycin inhibited ATP release from cultured astrocytes after ADP treatment GFAP and mTOR expression were detected with duallabeled fluorescence to evaluate the effect of ADP on the cultured astrocytes. The results showed that the expression of the GFAP increased about threefold (relative to naı¨ ve control) and expression of mTOR increased more than eight times (relative to naı¨ ve control) after exposure to 100 lM ADP for 24 h
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Fig. 1. Expression level of GFAP in the spinal cord increased gradually after nerve ligation. Chronic constriction of the sciatic nerve injury was performed on SD rats, (A) GFAP expression (brown DAB staining) was assessed through immunohistochemistry from the L4–6 dorsal spinal cord on day 1–21. (B) The mean intensity of GFAP staining (DAB staining, fold increase compared to naive controls) in CCI rats, determined by immunohistochemistry. Data are expressed as mean ± SEM, n = 6; ⁄P < 0.05, ⁄⁄⁄P < 0.001. Scale bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(Fig. 4A–C). We then verified whether ATP release was from astrocytes. The ATP concentration in astrocyte culture medium was increased significantly after being treated with 100 lM ADP for 24 h (37.71 ± 2.76 lM vs 1.10 ± 0.40 lM in the naı¨ ve control cells, P < 0.001). This increase was inhibited either by GDPbetaS or rapamycin (P < 0.001) (Fig. 4D). Rapamycin inhibited ATP release in the CSF of CCI rats To further investigate the effect of mTOR inhibitor on the activation of astrocytes, we evaluated the effect of rapamycin on the ATP concentration in the CSF of the CCI rats. The ATP level in the CSF of the CCI rats was significantly higher than that of naı¨ ve control rats (19.26 ± 0.56 lM vs 0.50 ± 0.11 lM, P < 0.001), which further increased in the CSF of the ADP-treated CCI rats (27.99 ± 1.26 lM vs 19.26 ± 0.56 lM in the CCI rats, P < 0.001). Intrathecal injection of rapamycin inhibited the ATP level increase significantly either in the CCI rats or ADP-treated CCI rats (P < 0.001) (Fig. 5).
Effect of rapamycin on the pain sensation of CCI rats The mechanical pain threshold and thermal withdraw latency of the ipsilateral foot were assessed to determine the effect of rapamycin on the pain sensation of CCI rats (Fig. 6). From day 7 after CCI surgery, rapamycin-treated CCI rats had a higher mechanical pain threshold (6.99 ± 1.06 g vs 4.62 ± 1.18 g on day 7, P < 0.01) and a higher thermal withdraw latency (17.53 ± 1.62 s vs 10.08 ± 2.10 s on day 7, P < 0.01) than CCI rats. There was no significant difference of mechanical pain threshold and thermal withdraw latency at each corresponding time point (P > 0.05) between the CCI rats and ADP-treated CCI rats except the thermal latency on day 3 (15.31 ± 1.89 s in CCI rats vs 9.79 ± 0.89 s in the ADP-treated CCI rats, P < 0.01). Rapamycin-treated CCI rats had a higher mechanical pain threshold (7.02 ± 1.28 g vs 3.86 ± 1.02 g at day 7, P < 0.01) and a higher thermal withdraw latency (15.81 ± 2.85 s vs 10.21 ± 0.76 s at day 7, P < 0.01) than CCI rats with ADP treatment only from day 7 after CCI surgery (Fig. 6).
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Fig. 2. Effects of administration of ADP and rapamycin on GFAP expression in CCI rats. The CCI rats received a daily intrathecal injection of 10 nmol ADP, pretreated with or without 0.01-nmol rapamycin from day 1 of CCI. Naı¨ ve control rats received same volume saline at the same time. (A) GFAP expression (brown DAB staining) in the L4–6 dorsal spinal cord was assessed by immunohistochemistry on day 14 after the first intrathecal injection. Up-regulation of GFAP expression levels in the spinal cord 14 days after sciatic nerve ligation was aggravated by ADP, which was attenuated by pretreated with 0.01-nmol rapamycin. (B) The mean staining intensity of GFAP in CCI rats, determined by immunohistochemistry. Data are expressed as mean ± SEM, n = 6; ⁄⁄⁄P < 0.001. Scale bar = 50 lm. Naı¨ ve contr: Naı¨ ve control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
DISCUSSION In the present study, astrocytes in the spinal dorsal horn were found to be converted to a reactive phenotype as a consequence of peripheral nerve injury. The pharmacological inhibition of mTOR reduced the spinal astrocyte activation and attenuated the neuropathic pain in rats. Furthermore, the inhibition of mTOR abolished the ATP release induced by exogenous ADP. These findings indicated that mTOR might be involved in neuropathic pain development, possibly through the activation of astrocytes induced by extracellular ATP/ ADP in the spinal dorsal horn in rats. Pain serves as a protective mechanism against injury. However, persistent nociceptive stimuli may develop into chronic pain, a pathological condition that disrupts the daily life of the patients (Zhuo, 2007). Although pain signaling is transmitted through neurons, accumulating evidence has implicated activation of spinal astrocytes in initiating and maintaining the development of chronic pain, especially neuropathic pain after peripheral nerve injury (Svensson and Brodin, 2010). Astrocytes express the glutamate transporters GLT-1 and GLAST (Perego et al., 2000), which are responsible for glutamate clearance from synaptic clefts and the extracellular space. The expression of these transporters were reduced after peripheral nerve injury, leading to impaired glutamate uptake and subsequent increase in excitatory synaptic transmission (Sung et al., 2003), which promotes spontaneous pain sensation and hypersensitivity to mechanical and thermal stimuli (Xin et al., 2009). In our study, increased intensity of GFAP staining was attributed primarily to hypertrophy and proliferation of astrocytes. The increase in GFAP staining in the spinal cord correlated well with the degree of pain. This is consistent with other studies (Romero-Sandoval et al., 2008), which established the strong association between GFAP expression and neuropathic pain in spinal cord.
ATP is the principal high-energy phosphate molecule that powers the cellular function (Yoshida et al., 2001). Release of ATP is caused by cell death or exocytosis from different cells (Praetorius and Leipziger, 2009). Extracellular ATP acts as a local mediator in an autocrine or paracrine manner within tissues to stimulate its receptors before its degradation, and this interaction induces physiological and immune responses in a wide spectrum of cell types (Khakh and Burnstock, 2009). Extracellular ATP binds to either the ligand-gated ion channel P2X receptors or the G protein-coupled P2Y receptors, and stimulates the phosphorylation of cell signaling molecules, including extracellular signal-regulated kinase (ERK) 1/2 (Chang et al., 2007) and p38 mitogen-activated protein kinase (MAPK) (Aimond et al., 2000). Recently, extracellular ATP has been found to induce electrophysiological and biological responses via P2X receptors or P2Y receptors on sensory neurons (Gerevich and Illes, 2004; Khakh and North, 2012), responsible for the pain in laboratory animals (Hamilton and McMahon, 2000). There is limited evidence for an effect of ADP, the major degradation product of ATP, on the pain signal transmission induced by peripheral nerve injury (Malin and Molliver, 2010). In our study, we found that exogenous ADP enhanced the astrocyte activation in the spinal dorsal horn and further increased pain sensitivity in the CCI rats. ADP is the main degradation product of ATP in extracellular space, and therefore our data indicated ADP may also play a role in chronic pain induction and maintenance. The mTOR signaling pathway regulates transcription, initiation of translation, and ribosome biosynthesis in response to multiple activating factors (Zoncu et al., 2011; Shimobayashi and Hall, 2014). However, whether the mTOR signaling pathway is involved in the regulation of pain and hyperalgesia remains incompletely understood. Recent studies demonstrated that astrocyte activation and proliferation are mediated by mTOR, which enhanced protein synthesis essential for this process
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Fig. 3. Effects of administration of ADP and rapamycin on mTOR expression in astrocytes of CCI rats. The CCI rats received daily intrathecal injection of 10 nmol ADP, pretreated with or without 0.01-nmol rapamycin from 1st day of CCI. Naı¨ ve control rats received same volume saline at the same time. After 14-day injection, the slices of L4–6 segments harvested from rats were used for immune-fluorescence staining. (A) Dual labeling of GFAP (red fluorescence) and mTOR (green fluorescence) in the L4–6 dorsal spinal cord on day 14 after first intrathecal injection. (B) The mean fluorescence intensity of GFAP determined by immune-fluorescence. (C) The mean fluorescence intensity of mTOR determined by immunofluorescence. Data are expressed as mean ± SEM, n = 6; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. Scale bar = 50 lm. Rap: rapamycin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(Banerjee et al., 2011a). The downstream mTOR effector proteins are crucial for translation of such signals, including eIF4E binding protein-1 (4EBP1), ribosomal protein S6 kinase (p70S6K), and eukaryotic elongation factor 2 (eEF2) (Codeluppi et al., 2009). In addition, mTOR pathway has been demonstrated to be involved in central and peripheral nociceptive sensitization (Tillu et al., 2012). Activity of spinal mTOR signaling pathways have been found to be responsible for the spinal neuronal plasticity and behavioral hypersensitivity caused by with nerve injury (Asante et al., 2010), and intrathecal injection of rapamycin has been shown to reduce neuropathic pain (Geranton et al., 2009). In our study, rapamycin, an mTOR inhibitor and GDPbetaS, a P2Y receptor antagonist, has significantly inhibited ATP release induced by exogenous ADP in cultured astrocytes. Our data have demonstrated that in the presence of rapamycin, exogenous ADP did not further up-regulate GFAP and mTOR expression in the
spinal cord (Fig. 3). This indicated that mTOR was involved in the signaling transduction induced by extracellular ADP, and inhibiting mTOR could directly abolish enhanced GFAP expression, which was not affected by additional administration of ADP. All these findings indicated that mTOR is involved in the signal transduction induced by extracellular ADP and might be modulated by P2Y receptors. As an important modulator in the physiology and pathology of the nervous system, mTOR has at least three upstream signaling molecules, AMPK, Akt and TSC (tuberous sclerosis complex) (Dalle Pezze et al., 2012; Kanno et al., 2012; Zhang et al., 2013). Further investigation is needed to identify which signal pathway connects the P2Y receptors to mTOR and participates in the ADP-induced astrocyte activation and other laboratory techniques should be considered to verify the findings in our studies. Early studies have shown that noxious stimulation or nerve injury leads to glial activation in the spinal dorsal
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Fig. 4. P2Y receptor antagonist, GDPbetaS and mTOR inhibitor rapamycin inhibited the release of ATP from the activated astrocytes after ADP exposure in vitro. The primary cultured rat spinal astrocytes exposed to 100 lM ADP with or not pretreated with GDPbetaS (100 lM) or rapamycin (10 nM). Cells treated with same volume of 0.9% saline was considered as vehicle control. (A) Multiple labeling of GFAP (red fluorescence), mTOR (green fluorescence) and nucleus (blue fluorescence) in cultured rat spinal astrocytes. (B) The intensity of GFAP red fluorescence compared with naı¨ ve control. (C) The intensity of mTOR green fluorescence compared with naı¨ ve control. (D) ATP concentration in the medium of cultured spinal astrocytes assessed by a firefly luciferase ATP assay kit. The increase of the ATP concentration in culture medium of astrocytes was suppressed by GDPbetaS or rapamycin. Data are expressed as mean ± SEM, n = 4; ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. Scale bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. mTOR inhibitor rapamycin inhibited ATP release in the CSF of CCI rats. The CCI rats received daily intrathecal injection of 10 nmol ADP, pre-treated with or without 0.01-nmol rapamycin. The ATP concentration in cerebral spinal fluid (CSF) in CCI rats assessed by a firefly luciferase ATP assay kit. Increase of ATP release was abolished by rapamycin. Data are expressed as mean ± SEM, n = 6; ⁄⁄⁄P < 0.001.
horn, which in turn causes an amplification of the response to sensory inputs (Tsuda et al., 2003). It is now accepted in basic science that inhibiting the activation of spinal glia is an effective method to treat chronic pain induced by peripheral nerve injury. In this study, we found that exogenous ADP increased high level of astrocyte activation (Fig. 2) and the development of pain (Fig. 6), which indicated that ADP was a potent modulator in the development of hyperalgesia. In addition,
rapamycin administration via an intrathecal catheter significantly inhibited astrocyte activation. This would help us to further understand the action of rapamycin in analgesia. It was reported that rapamycin could reduce the level of chronic pain induced by ligation of peripheral nerves by acting on an mTOR-positive subset of A-nociceptors, laminaI neuronal projections and glial cells (Tillu et al., 2012). We have reported that, rapamycin suppressed the astrocyte activation and ATP release induced by exogenous ADP in vitro. In the central nervous system, ATP is released from not only astrocytes but also neurons and other cells (Rozanski et al., 2013). In the present study, rapamycin has shown a beneficial effect by preventing the induction and progression of the neuropathic pain associated with ATP or ADP. Intrathecal injection may provide either temporal or permanent pain relief, which allows other treatments, such as physical therapy, to be more effective. Much less dose is needed with intrathecal injection than systematic administration and the side effects of drugs were significantly reduced (Hayek and Hanes, 2014). Rapamycin could easily cross the blood–brain barrier and move into spinal space, in addition, it was reported that brain rapamycin levels exponentially correlated with blood rapamycin levels after intraperitoneal administration (Banerjee et al., 2011b). Given the clinical availability of rapamycin and the intrathecal delivery techniques, the long-term intrathecal treatment with rapamycin might be predicted to be feasible in neuropathic pain management, this certainly warrants further investigations in future studies.
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Fig. 6. Intrathecal administration of rapamycin attenuated the mechanical allodynia and thermal sensitivity of CCI rats. The CCI rats received daily intrathecal injection of 10 nmol ADP, pretreated with or without 0.01-nmol rapamycin from day 1 of CCI, and naı¨ ve control rats received same volume of 0.9% saline at the same time. (A) The mechanical allodynia of CCI rats on day 3–21, Intrathecal administration of 0.01-nmol rapamycin reversed the CCI-induced decrease in mechanical threshold. (B) Thermal sensitivity of CCI rats assessed by Hargreaves paw withdrawal test. Intrathecal administration of 0.01-nmol rapamycin reversed the CCI-induced decrease in thermal hypersensitivity. Data are expressed as mean ± SEM, n = 6. ⁄⁄P < 0.01 compared with naı¨ ve control; ##P < 0.01 compared with the rats in CCI group on the same day.
CONCLUSIONS Our study demonstrated that mTOR signaling pathway is involved in the neuropathic pain development, which is likely associated with the release of ATP. Administration of rapamycin suppressed spinal astrocyte activation and ATP release, and thereby displayed powerful antinociceptive effects. The present study may suggest that targeting mTOR signaling pathway in the spinal cord might be a promising therapeutic approach to alleviate the chronic pain.
COMPETING INTERESTS Authors declare no competing interests. Acknowledgments—This research is supported by grants from the National Natural Science Foundation of China (No. 31171069) and Braun Anesthesia Scientific Research Fund (No. BBF-2012-09).
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(Accepted 13 June 2014) (Available online 27 June 2014)
mTOR PATHWAY IS INVOLVED IN ADP-EVOKED ASTROCYTE ACTIVATION AND ATP RELEASE IN THE SPINAL DORSAL HORN IN A RAT NEUROPATHIC PAIN MODEL J. CUI, a W. HE, b B. YI, a H. ZHAO, c K. LU, a* H. RUAN b* AND D. MA c
Key words: adenosine triphosphate, adenosine diphosphate, astrocyte, mammalian target of rapamycin, neuropathic pain.
a Department of Anaesthesiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China b Department of Neurobiology, Chongqing Key Laboratory of Neurobiology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China
INTRODUCTION Neuropathic pain often develops when nerves are damaged through diabetes, surgery, infection and physical injury, and does not resolve upon the healing of the overt damaged tissue (Costigan et al., 2009). The current clinical management of neuropathic pain is not optimal and in fact, patients are relatively unresponsive to most available treatments, such as anticonvulsive, antidepressant and even opioids (Hearn et al., 2014). Mounting evidence has indicated that neuropathic pain is induced by the aberrant excitability of dorsal horn neurons evoked by peripheral sensory inputs (Costigan et al., 2009). Apart from molecular and cellular alterations in primary sensory neurons and in the spinal dorsal horn after nerve injury, recent studies have identified that spinal astrocyte activation, termed the reactive astrogliosis (Karimi-Abdolrezaee and Billakanti, 2012), enhances the production of various mediators, including proinflammatory cytokines (Brambilla et al., 2005) and brain-derived neurotrophic factor (Zhang et al., 2011), which promotes the hyperexcitability of dorsal horn neurons (Gwak and Hulsebosch, 2009). Reactive astrogliosis has been shown as a reflection of the severity of peripheral nerve injury and is characterized by cellular hypertrophy, increased glial fibrillary acidic protein (GFAP) expression and cellular proliferation (Cirillo et al., 2012). Molecular mechanisms that control the initiation of the development of the reactive astrogliosis are, nonetheless, poorly understood. Recently, studies have demonstrated that the extracellular ATP could act as a crucial neuromodulator on glial cell activation in the central nervous system and plays an important role in the neuropathic pain developed from peripheral nerve injury (Jakobsson, 2010). The mechanism of ATP release from certain cells and the effects of its degradation (ADP and AMP) on the induction of glial cell activation have recently attracted more attention in pain research (Burnstock, 2012). P2X ion channels and P2Y G protein-coupled receptors have been identified on neural cells, which could be activated by extracellular ATP (Burnstock et al., 2011). ADP is the primary enzymatic breakdown product in extracellular space and only contributes to the activation of P2Y receptors (Malin and
c
Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea & Westminster Hospital, London, UK
Abstract—Background: ATP/ADP-evoked spinal astrocyte activation plays a vital role in the development of neuropathic pain. We aim to investigate the role of mammalian target of rapamycin (mTOR) pathway on the spinal astrocyte activation in the neuropathic pain development in rats. Methods: Sprague Dawley (SD) rats were subjected to chronic constriction of the sciatic nerve (CCI). Rapamycin or ADP was intrathecally injected daily to explore their effects on spinal astrocyte activation and pain development. Expression of glial fibrillary acidic protein (GFAP) and mTOR in the spinal dorsal horn was assessed by immunohistochemistry. Von Frey hairs and Hargreaves paw withdrawal test were conducted to evaluate mechanical allodynia and thermal sensitivity, respectively. Firefly luciferase ATP assay was used to assess the change of ATP level in cerebrospinal fluid (CSF) and medium of cultured astrocytes. Results: GFAP expression was enhanced in the ipsilateral spinal dorsal horn from day 3 after surgery. GFAP and mTOR expression in the rat spinal dorsal horn on post-surgical day 14 was enhanced by daily intrathecal injection of ADP, which was inhibited by rapamycin. Rapamycin decreased lower mechanical pain threshold and the thermal withdrawal latency. Intrathecal injection of ADP enhanced the ATP release, which was partially inhibited by rapamycin. Study of cultured astrocytes indicated that ATP could be released from astrocytes. Conclusion: Our data demonstrated that ADP enhanced neuropathic pain in CCI rats, which was inhibited by rapamycin. This study indicates that targeting mTOR pathway could serve as a novel therapeutic strategy in neuropathic pain management. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding authors. Tel/fax: +86-23-68754197 (K. Lu), +8623-68753672 (H. Ruan). E-mail addresses: [email protected] (K. Lu), [email protected] (H. Ruan). Abbreviations: AMPK, AMP-activated protein kinase; CCI, chronic constriction injury model; CSF, cerebrospinal fluid; GFAP, glial fibrillary acidic protein; mTOR, mammalian target of rapamycin; SD, Sprague Dawley; vFh, Von Frey hairs. http://dx.doi.org/10.1016/j.neuroscience.2014.06.030 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 395
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Molliver, 2010). However, it remains unclear whether ADP could activate glial cells and induce neuropathic pain after peripheral nerve injury. Mammalian target of rapamycin (mTOR) pathway has been found to play a vital role in controlling local protein synthesis in neuronal axons and dendrites, as well as in modulating memory processes and plasticity (Takei et al., 2004). Rapamycin, an inhibitor of mTOR, has been reported to inhibit the activation of glial cells in the spinal cord and brain (Norsted Gregory et al., 2013; Oubrahim et al., 2013). Furthermore, it has been shown that neuropathic pain in mice was reduced through intraperitoneal injection of rapamycin (Obara et al., 2011). In addition, mTOR pathway plays an important role in transmitting the signal from G-coupled protein to mitochondria, which regulates many cellular activities such as cell proliferation and cytokine release (Villa-Cuesta et al., 2014). Recently, mTOR has become a therapeutic target in the activation of neural cells (Morgan-Warren et al., 2013). Given the pivotal role of astrocytes in promoting and sustaining chronic neuropathic pain, targeting astrocytes could reveal novel therapies for the clinical management. We have previously demonstrated that ADP-evoked extracellular ATP accumulation around astrocytes is mediated by P2Y receptors and AMPactivated protein kinase (AMPK) (Cui et al., 2011). AMPK and mTOR signaling pathways are associated with the ATP synthesis and consumption in cytoplasm and targeting both pathways has been shown to be beneficial for the treatment with pain (Melemedjian et al., 2011; Obara et al., 2011). In the present study, we aim to explore the role of the mTOR signaling pathway in the ATP release from the astrocytes through using the rat chronic constriction injury model (CCI), a well-established model of neuropathic pain (Bennett et al., 2003). Furthermore, the painrelieving effect of rapamycin on ATP/ADP-mediated neuropathic pain was also evaluated.
EXPERIMENTAL PROCEDURE Animals and surgery Adult female Sprague Dawley (SD) rats (200–250 g) were used for establishing neuropathic pain model (Tall et al., 2001) and post-natal SD rats (1–3 days old) were used to prepare astrocyte culture of the spinal cord. The rats were provided by the Center of Laboratory Animals of Third Military Medical University, Chongqing, China. All experiment protocols were approved by the Ethics Committee of Third Military Medical University. Efforts have been made to reduce animal suffering and minimize animal number used. Animals were housed in plastic cages with free access to food and water under a 12-h light/dark cycle. Intrathecal catheters were implanted as previously described (Yang et al., 2012). Briefly, adult female SD rats were anesthetized with 3.5% chloral hydrate (10 ml/kg, i.p.). The L4–L5 vertebra of the rat was opened on the dorsal side, and then the L5 spinouts process was removed. A polyethylene catheter (PE-10, BD, USA) was implanted to a depth of about 1.0 cm from the incision between the gaps of L4 and L5 vertebra and the surgical wound was sutured. The other end of the catheter was
exited at the back neck of the rat via a subcutaneous tunnel and was secured in situ. The correct intrathecal catheter positioning was confirmed with the paralysis of the bilateral hind limbs after 2% lidocaine (10 ll) injection through the catheter. Those with success of catheter positioning were allowed for 3–5-day recovery before the right sciatic nerve ligation surgery (Bennett and Xie, 1988). Briefly, the right common sciatic nerve branches (close to the trifurcation into the sural, peroneal and tibial nerve) were exposed and tied with 4–0 cotton sutures under chloral hydrate (3.5%) anesthesia. After intrathecal catheter was implanted successfully, rats without any other surgeries served as the naı¨ ve control. Rats with right sciatic nerve exposure without ligation served as the sham controls. Animals were used for experiments for up to 21 days after CCI surgery. Drug administration Rats were administered intrathecally with 10-ll rapamycin (100 nM, Merck Millipore, USA) or saline (10 ll as vehicle control), and 15 min later, 10 ll ADP (1 mM, Sigma, USA) was administered through the same route. The drugs were used once a day immediately after nerve injury until post-surgical day 14. Mechanical and thermal sensitivity measurement Von Frey hairs test (vFh) with bending forces at a range of 0.3–20.3 g was used to assess mechanical allodynia (He et al., 2012). The test began with the 4.10-g vFh (the middle of the filament series). The ventral surface of each hind paw was subjected to the filament for 4–6 s. If the animal lifted the hind paw quickly, it was recorded as a positive paw withdrawal response. The up-down method was used to determine the 50% paw-withdrawal mechanical threshold (PWMT). Hargreaves paw withdrawal was used to assess thermal sensitivity. It measured the withdrawal latency from a radiant heat source applied on the proximal half of the plantar surface of each hind paw (Han et al., 2011). The paw-withdrawal thermal latency (PWTL) was recorded as the threshold of thermal sensitivity. Each hind paw was tested five times in every 5 min. Immunohistochemistry Under terminated anesthesia (chloral hydrate 350 mg/kg, i.p.), rats were perfused intracardially with 4% paraformaldehyde. Spinal cords were quickly removed from rats and fixed overnight. After dehydration and cryoprotection for 48 h in 30% sucrose at 4 °C, the L4–6 segments of the frozen spinal cords were sectioned into 16-lm slices. After inhibition of endogenous peroxidase with 3% H2O2 for 15 min (this process will be omitted for immune-fluorescence staining), the slices were incubated with 10% goat serum for 1 h and then incubated with primary rabbit anti-rat mTOR (1:250, Millipore, USA) antibody and mouse anti-rat GFAP antibody (1:300, Sigma, USA) overnight at 4 °C. For ex vivo DAB staining, the antibody was visualized with a nickel-intensified DAB chromogen and the images were
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obtained with a microscope (Leica, Wetzlar, Germany). For the immune-fluorescence, the section was incubated with Cy3-conjugated goat anti-rabbit antibody (1:300, Jackson Immuno-Research, West Grove, PA, USA) and FITC-conjugated goat anti-mouse antibody (1:300, Jackson Immuno-Research) for 1 h. The nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, 1:1000, Sigma, USA) and examined with a laser scanning confocal microscope (Olympus FLV1000, Germany). Five randomly selected spinal sections were chosen for each animal, and the mean intensity of each section was analyzed with Image J software (NIH, US National Institutes of Health, Bethesda, MD). Data were expressed as the percentage relative to that of the naı¨ ve controls. Spinal astrocyte culture The primary culture of dorsal spinal cord astrocytes was prepared (Zeng et al., 2008). Briefly, postnatal SD rats (1–3 days old) were killed by decapitation and spinal cords were removed quickly. Meninges were removed under dissecting microscope and L1–L6 sections were harvested. The spinal cords were sectioned and then dissociated with 0.25% trypsin (Sigma, USA) for 15 min at 37 °C. The cells were cultured in the DMEM-F12 medium supplemented with 10% fetal calf serum and 50U/ml penicillin, 5 lg/ml streptomycin (Sigma–Aldrich, USA) for 10 days and then centrifuged at 450 rpm for 12 h to remove microglia and neuron. The quality of astrocyte culture was confirmed through using GFAP staining and astrocyte purity over 95% was used. ATP level assessment The ATP levels in rat cerebrospinal fluid (CSF) and cell culture medium were assessed through the firefly luciferase ATP assay kit (Beyotime, China) as described previously (Cui et al., 2011). Briefly, astrocytes cultured in 6-well plates were treated with 100 lM GDPbetaS (antagonist of P2Y receptors) or 10 nM rapamycin for 15 min and then exposed to 100 lM ADP for additional 24 h. Culture medium was collected from the astrocyte culture medium and kept at 80 °C before detection. Rat CSF was collected through a 22-G needle punctured into the L4–L5 spinal space of rats close to intrathecal catheter. 150-ll CSF or 500-ll culture medium was placed in 1.5-ml Eppendorf tubes and then centrifuged at 12,000g for 5 min at 4 °C. After centrifugation, 100 ll of the supernatant was mixed with 100 ll of ATP detection solution at its working dilution in a 1.5-ml Eppendorf tube. Luminance (RLU) was measured using a GloMax 20/20 luminometer (Promega, USA) and the concentration of ATP was measured. Statistical analysis All data were presented as mean ± SEM. Data were collected from at least four independent experiments (n P 4) and were analyzed statistically with a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc tests if multiple
comparisons were necessary (SPSS16.0, SPSS Inc., Chicago, IL, USA). A P-value of 0.05 was considered as statistically significant.
RESULTS Astrocytes were activated in the spinal dorsal horn after CCI The GFAP expression, a typical marker for astrocyte activation, was assessed in the ipsilateral spinal dorsal horn in the rats after CCI through immunohistochemistry. The expression of GFAP was enhanced gradually in the ipsilateral spinal dorsal horn from day 3 after the partial sciatic nerve ligation of rats when compared with that of controls (Fig. 1A). The intensity of GFAP staining was increased to the highest level 14 days after CCI surgery (Fig. 1B). ADP further enhanced astrocyte activation which was attenuated by rapamycin administration To investigate the effects of ADP and rapamycin on the activation of spinal astrocytes in CCI rats, rapamycin and ADP were injected into the spinal space daily through the intrathecal tube embedded in advance. The increase in GFAP expression that was observed in the rat spinal dorsal horn on the day 14 after the sciatic nerve ligation operation was enhanced by injection of 10 nmol ADP (P < 0.01, 472.73 ± 36.39% vs 312.65 ± 35.01% in CCI rats without ADP treatment). This high expression of GFAP induced by ADP was inhibited by pre-treatment with 0.01-nmol rapamycin (P < 0.001, 198.36 ± 25.41% vs 472.73 ± 36.39% in the ADP-treated CCI rats) (Fig. 2). Enhanced expression of mTOR was attenuated by rapamycin on the spinal astrocytes of CCI rats Expression of mTOR on the spinal astrocytes of CCI rats was assessed with a dual-labeling immune-fluorescence assay. mTOR and GFAP fluorescence staining were found to be co-localized (Fig. 3A, B), indicating enhanced expression of mTOR in activated astrocytes. The expression of mTOR was significantly increased on day 14 after CCI operation (137.61 ± 3.51% relative to control), which was enhanced by intrathecal injection with ADP (P < 0.01, 178.51 ± 18.05% vs 137.61 ± 3.51% in CCI rats), furthermore, this increase in expression could be suppressed by intrathecal injection of rapamycin (P < 0.001, 128.56 ± 8.67% vs 178.51 ± 18.05% in CCI rats treated with ADP) (Fig. 3C). Rapamycin inhibited ATP release from cultured astrocytes after ADP treatment GFAP and mTOR expression were detected with duallabeled fluorescence to evaluate the effect of ADP on the cultured astrocytes. The results showed that the expression of the GFAP increased about threefold (relative to naı¨ ve control) and expression of mTOR increased more than eight times (relative to naı¨ ve control) after exposure to 100 lM ADP for 24 h
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Fig. 1. Expression level of GFAP in the spinal cord increased gradually after nerve ligation. Chronic constriction of the sciatic nerve injury was performed on SD rats, (A) GFAP expression (brown DAB staining) was assessed through immunohistochemistry from the L4–6 dorsal spinal cord on day 1–21. (B) The mean intensity of GFAP staining (DAB staining, fold increase compared to naive controls) in CCI rats, determined by immunohistochemistry. Data are expressed as mean ± SEM, n = 6; ⁄P < 0.05, ⁄⁄⁄P < 0.001. Scale bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(Fig. 4A–C). We then verified whether ATP release was from astrocytes. The ATP concentration in astrocyte culture medium was increased significantly after being treated with 100 lM ADP for 24 h (37.71 ± 2.76 lM vs 1.10 ± 0.40 lM in the naı¨ ve control cells, P < 0.001). This increase was inhibited either by GDPbetaS or rapamycin (P < 0.001) (Fig. 4D). Rapamycin inhibited ATP release in the CSF of CCI rats To further investigate the effect of mTOR inhibitor on the activation of astrocytes, we evaluated the effect of rapamycin on the ATP concentration in the CSF of the CCI rats. The ATP level in the CSF of the CCI rats was significantly higher than that of naı¨ ve control rats (19.26 ± 0.56 lM vs 0.50 ± 0.11 lM, P < 0.001), which further increased in the CSF of the ADP-treated CCI rats (27.99 ± 1.26 lM vs 19.26 ± 0.56 lM in the CCI rats, P < 0.001). Intrathecal injection of rapamycin inhibited the ATP level increase significantly either in the CCI rats or ADP-treated CCI rats (P < 0.001) (Fig. 5).
Effect of rapamycin on the pain sensation of CCI rats The mechanical pain threshold and thermal withdraw latency of the ipsilateral foot were assessed to determine the effect of rapamycin on the pain sensation of CCI rats (Fig. 6). From day 7 after CCI surgery, rapamycin-treated CCI rats had a higher mechanical pain threshold (6.99 ± 1.06 g vs 4.62 ± 1.18 g on day 7, P < 0.01) and a higher thermal withdraw latency (17.53 ± 1.62 s vs 10.08 ± 2.10 s on day 7, P < 0.01) than CCI rats. There was no significant difference of mechanical pain threshold and thermal withdraw latency at each corresponding time point (P > 0.05) between the CCI rats and ADP-treated CCI rats except the thermal latency on day 3 (15.31 ± 1.89 s in CCI rats vs 9.79 ± 0.89 s in the ADP-treated CCI rats, P < 0.01). Rapamycin-treated CCI rats had a higher mechanical pain threshold (7.02 ± 1.28 g vs 3.86 ± 1.02 g at day 7, P < 0.01) and a higher thermal withdraw latency (15.81 ± 2.85 s vs 10.21 ± 0.76 s at day 7, P < 0.01) than CCI rats with ADP treatment only from day 7 after CCI surgery (Fig. 6).
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Fig. 2. Effects of administration of ADP and rapamycin on GFAP expression in CCI rats. The CCI rats received a daily intrathecal injection of 10 nmol ADP, pretreated with or without 0.01-nmol rapamycin from day 1 of CCI. Naı¨ ve control rats received same volume saline at the same time. (A) GFAP expression (brown DAB staining) in the L4–6 dorsal spinal cord was assessed by immunohistochemistry on day 14 after the first intrathecal injection. Up-regulation of GFAP expression levels in the spinal cord 14 days after sciatic nerve ligation was aggravated by ADP, which was attenuated by pretreated with 0.01-nmol rapamycin. (B) The mean staining intensity of GFAP in CCI rats, determined by immunohistochemistry. Data are expressed as mean ± SEM, n = 6; ⁄⁄⁄P < 0.001. Scale bar = 50 lm. Naı¨ ve contr: Naı¨ ve control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
DISCUSSION In the present study, astrocytes in the spinal dorsal horn were found to be converted to a reactive phenotype as a consequence of peripheral nerve injury. The pharmacological inhibition of mTOR reduced the spinal astrocyte activation and attenuated the neuropathic pain in rats. Furthermore, the inhibition of mTOR abolished the ATP release induced by exogenous ADP. These findings indicated that mTOR might be involved in neuropathic pain development, possibly through the activation of astrocytes induced by extracellular ATP/ ADP in the spinal dorsal horn in rats. Pain serves as a protective mechanism against injury. However, persistent nociceptive stimuli may develop into chronic pain, a pathological condition that disrupts the daily life of the patients (Zhuo, 2007). Although pain signaling is transmitted through neurons, accumulating evidence has implicated activation of spinal astrocytes in initiating and maintaining the development of chronic pain, especially neuropathic pain after peripheral nerve injury (Svensson and Brodin, 2010). Astrocytes express the glutamate transporters GLT-1 and GLAST (Perego et al., 2000), which are responsible for glutamate clearance from synaptic clefts and the extracellular space. The expression of these transporters were reduced after peripheral nerve injury, leading to impaired glutamate uptake and subsequent increase in excitatory synaptic transmission (Sung et al., 2003), which promotes spontaneous pain sensation and hypersensitivity to mechanical and thermal stimuli (Xin et al., 2009). In our study, increased intensity of GFAP staining was attributed primarily to hypertrophy and proliferation of astrocytes. The increase in GFAP staining in the spinal cord correlated well with the degree of pain. This is consistent with other studies (Romero-Sandoval et al., 2008), which established the strong association between GFAP expression and neuropathic pain in spinal cord.
ATP is the principal high-energy phosphate molecule that powers the cellular function (Yoshida et al., 2001). Release of ATP is caused by cell death or exocytosis from different cells (Praetorius and Leipziger, 2009). Extracellular ATP acts as a local mediator in an autocrine or paracrine manner within tissues to stimulate its receptors before its degradation, and this interaction induces physiological and immune responses in a wide spectrum of cell types (Khakh and Burnstock, 2009). Extracellular ATP binds to either the ligand-gated ion channel P2X receptors or the G protein-coupled P2Y receptors, and stimulates the phosphorylation of cell signaling molecules, including extracellular signal-regulated kinase (ERK) 1/2 (Chang et al., 2007) and p38 mitogen-activated protein kinase (MAPK) (Aimond et al., 2000). Recently, extracellular ATP has been found to induce electrophysiological and biological responses via P2X receptors or P2Y receptors on sensory neurons (Gerevich and Illes, 2004; Khakh and North, 2012), responsible for the pain in laboratory animals (Hamilton and McMahon, 2000). There is limited evidence for an effect of ADP, the major degradation product of ATP, on the pain signal transmission induced by peripheral nerve injury (Malin and Molliver, 2010). In our study, we found that exogenous ADP enhanced the astrocyte activation in the spinal dorsal horn and further increased pain sensitivity in the CCI rats. ADP is the main degradation product of ATP in extracellular space, and therefore our data indicated ADP may also play a role in chronic pain induction and maintenance. The mTOR signaling pathway regulates transcription, initiation of translation, and ribosome biosynthesis in response to multiple activating factors (Zoncu et al., 2011; Shimobayashi and Hall, 2014). However, whether the mTOR signaling pathway is involved in the regulation of pain and hyperalgesia remains incompletely understood. Recent studies demonstrated that astrocyte activation and proliferation are mediated by mTOR, which enhanced protein synthesis essential for this process
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Fig. 3. Effects of administration of ADP and rapamycin on mTOR expression in astrocytes of CCI rats. The CCI rats received daily intrathecal injection of 10 nmol ADP, pretreated with or without 0.01-nmol rapamycin from 1st day of CCI. Naı¨ ve control rats received same volume saline at the same time. After 14-day injection, the slices of L4–6 segments harvested from rats were used for immune-fluorescence staining. (A) Dual labeling of GFAP (red fluorescence) and mTOR (green fluorescence) in the L4–6 dorsal spinal cord on day 14 after first intrathecal injection. (B) The mean fluorescence intensity of GFAP determined by immune-fluorescence. (C) The mean fluorescence intensity of mTOR determined by immunofluorescence. Data are expressed as mean ± SEM, n = 6; ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. Scale bar = 50 lm. Rap: rapamycin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(Banerjee et al., 2011a). The downstream mTOR effector proteins are crucial for translation of such signals, including eIF4E binding protein-1 (4EBP1), ribosomal protein S6 kinase (p70S6K), and eukaryotic elongation factor 2 (eEF2) (Codeluppi et al., 2009). In addition, mTOR pathway has been demonstrated to be involved in central and peripheral nociceptive sensitization (Tillu et al., 2012). Activity of spinal mTOR signaling pathways have been found to be responsible for the spinal neuronal plasticity and behavioral hypersensitivity caused by with nerve injury (Asante et al., 2010), and intrathecal injection of rapamycin has been shown to reduce neuropathic pain (Geranton et al., 2009). In our study, rapamycin, an mTOR inhibitor and GDPbetaS, a P2Y receptor antagonist, has significantly inhibited ATP release induced by exogenous ADP in cultured astrocytes. Our data have demonstrated that in the presence of rapamycin, exogenous ADP did not further up-regulate GFAP and mTOR expression in the
spinal cord (Fig. 3). This indicated that mTOR was involved in the signaling transduction induced by extracellular ADP, and inhibiting mTOR could directly abolish enhanced GFAP expression, which was not affected by additional administration of ADP. All these findings indicated that mTOR is involved in the signal transduction induced by extracellular ADP and might be modulated by P2Y receptors. As an important modulator in the physiology and pathology of the nervous system, mTOR has at least three upstream signaling molecules, AMPK, Akt and TSC (tuberous sclerosis complex) (Dalle Pezze et al., 2012; Kanno et al., 2012; Zhang et al., 2013). Further investigation is needed to identify which signal pathway connects the P2Y receptors to mTOR and participates in the ADP-induced astrocyte activation and other laboratory techniques should be considered to verify the findings in our studies. Early studies have shown that noxious stimulation or nerve injury leads to glial activation in the spinal dorsal
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Fig. 4. P2Y receptor antagonist, GDPbetaS and mTOR inhibitor rapamycin inhibited the release of ATP from the activated astrocytes after ADP exposure in vitro. The primary cultured rat spinal astrocytes exposed to 100 lM ADP with or not pretreated with GDPbetaS (100 lM) or rapamycin (10 nM). Cells treated with same volume of 0.9% saline was considered as vehicle control. (A) Multiple labeling of GFAP (red fluorescence), mTOR (green fluorescence) and nucleus (blue fluorescence) in cultured rat spinal astrocytes. (B) The intensity of GFAP red fluorescence compared with naı¨ ve control. (C) The intensity of mTOR green fluorescence compared with naı¨ ve control. (D) ATP concentration in the medium of cultured spinal astrocytes assessed by a firefly luciferase ATP assay kit. The increase of the ATP concentration in culture medium of astrocytes was suppressed by GDPbetaS or rapamycin. Data are expressed as mean ± SEM, n = 4; ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. Scale bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. mTOR inhibitor rapamycin inhibited ATP release in the CSF of CCI rats. The CCI rats received daily intrathecal injection of 10 nmol ADP, pre-treated with or without 0.01-nmol rapamycin. The ATP concentration in cerebral spinal fluid (CSF) in CCI rats assessed by a firefly luciferase ATP assay kit. Increase of ATP release was abolished by rapamycin. Data are expressed as mean ± SEM, n = 6; ⁄⁄⁄P < 0.001.
horn, which in turn causes an amplification of the response to sensory inputs (Tsuda et al., 2003). It is now accepted in basic science that inhibiting the activation of spinal glia is an effective method to treat chronic pain induced by peripheral nerve injury. In this study, we found that exogenous ADP increased high level of astrocyte activation (Fig. 2) and the development of pain (Fig. 6), which indicated that ADP was a potent modulator in the development of hyperalgesia. In addition,
rapamycin administration via an intrathecal catheter significantly inhibited astrocyte activation. This would help us to further understand the action of rapamycin in analgesia. It was reported that rapamycin could reduce the level of chronic pain induced by ligation of peripheral nerves by acting on an mTOR-positive subset of A-nociceptors, laminaI neuronal projections and glial cells (Tillu et al., 2012). We have reported that, rapamycin suppressed the astrocyte activation and ATP release induced by exogenous ADP in vitro. In the central nervous system, ATP is released from not only astrocytes but also neurons and other cells (Rozanski et al., 2013). In the present study, rapamycin has shown a beneficial effect by preventing the induction and progression of the neuropathic pain associated with ATP or ADP. Intrathecal injection may provide either temporal or permanent pain relief, which allows other treatments, such as physical therapy, to be more effective. Much less dose is needed with intrathecal injection than systematic administration and the side effects of drugs were significantly reduced (Hayek and Hanes, 2014). Rapamycin could easily cross the blood–brain barrier and move into spinal space, in addition, it was reported that brain rapamycin levels exponentially correlated with blood rapamycin levels after intraperitoneal administration (Banerjee et al., 2011b). Given the clinical availability of rapamycin and the intrathecal delivery techniques, the long-term intrathecal treatment with rapamycin might be predicted to be feasible in neuropathic pain management, this certainly warrants further investigations in future studies.
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Fig. 6. Intrathecal administration of rapamycin attenuated the mechanical allodynia and thermal sensitivity of CCI rats. The CCI rats received daily intrathecal injection of 10 nmol ADP, pretreated with or without 0.01-nmol rapamycin from day 1 of CCI, and naı¨ ve control rats received same volume of 0.9% saline at the same time. (A) The mechanical allodynia of CCI rats on day 3–21, Intrathecal administration of 0.01-nmol rapamycin reversed the CCI-induced decrease in mechanical threshold. (B) Thermal sensitivity of CCI rats assessed by Hargreaves paw withdrawal test. Intrathecal administration of 0.01-nmol rapamycin reversed the CCI-induced decrease in thermal hypersensitivity. Data are expressed as mean ± SEM, n = 6. ⁄⁄P < 0.01 compared with naı¨ ve control; ##P < 0.01 compared with the rats in CCI group on the same day.
CONCLUSIONS Our study demonstrated that mTOR signaling pathway is involved in the neuropathic pain development, which is likely associated with the release of ATP. Administration of rapamycin suppressed spinal astrocyte activation and ATP release, and thereby displayed powerful antinociceptive effects. The present study may suggest that targeting mTOR signaling pathway in the spinal cord might be a promising therapeutic approach to alleviate the chronic pain.
COMPETING INTERESTS Authors declare no competing interests. Acknowledgments—This research is supported by grants from the National Natural Science Foundation of China (No. 31171069) and Braun Anesthesia Scientific Research Fund (No. BBF-2012-09).
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(Accepted 13 June 2014) (Available online 27 June 2014)