Effects of dexmedetomidine on P2X4Rs, p38-MAPK and BDNF in spinal microglia in rats with spared nerve injury.

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Effects of dexmedetomidine on P2X4Rs, p38-MAPK and BDNF in spinal microglia in rats with spared nerve injury Tian-tian Zhou1, Jing-ru Wu1, Zi-yang Chen, Zhen-xiu Liu, Bei Miaon Jiangsu Province Key Laboratory of Anaesthesiology, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China

art i cle i nfo

ab st rac t

Article history:

Microglia in the spinal cord is evidenced to play a crucial role in neuropathic pain. Spinal

Accepted 15 April 2014

P2X4 receptors (P2X4Rs), which are mainly expressed in microglia, have been investigated for their roles in neuropathic pain. Dexmedetomidine (DEX), a highly selective agonist of α2-adrenergic receptors, is clinically applied to sedation and analgesia. Despite the

Keywords:

proposed mechanisms underlying DEX-induced analgesia, the possible interactions

Dexmedetomidine

between DEX and P2X4Rs at a molecular level have not been elucidated. We designated

Neuropathic pain

the spared nerve injury (SNI) to establish the neuropathic pain model. Mechanical paw

Microglia

withdrawal threshold (MWT) was measured to evaluate the sensitivity of neuropathic pain

Spinal cord

in rats. MWT was significantly decreased in SNI rats versus control rats. Expressions of

P2X4Rs

spinal P2X4Rs, phosphorylated p38-mitogen-activated protein kinase (p-p38-MAPK) and

p-p38-MAPK

brain-derived neurotrophic factor (BDNF) were upregulated in SNI rats. Immunofluores-

BDNF

cence assay indicated higher densities of microglia and P2X4Rs, which appeared yellow in colour, suggesting they were co-labelled. Intraperitoneal injections of DEX 40 μg/kg for 14 consecutive days markedly reversed the SNI-induced decline of MWT; the activation of microglia was markedly inhibited; in addition, the protein expressions of P2X4Rs, p-p38MAPK and BDNF were significantly downregulated. Thus, DEX could attenuate the neuropathic pain in SNI rats, of which the mechanism might be related to the downexpressed P2X4Rs, p-p38 and BDNF in microglia of spinal dorsal horn. & 2014 Elsevier B.V. All rights reserved.

Abbreviations: BDNF,

brain-derived neurotrophic factor; CNS,

calcium binding adaptor molecule 1; IP, MAPK,

intraperitoneal; MWT,

p38-mitogen-activated protein kinase; p-p38-MAPK,

central nervous system; DEX,

dexmedetomidine; Iba-1,

mechanical paw withdrawal threshold; P2X4Rs,

ionised

P2X4 receptors; p38-

phosphorylation of p38-mitogen-activated protein kinase; SNI,

spared

nerve injury; SD, Sprague-Dawley n Corresponding author. Fax: þ86 516 8326 2691. E-mail addresses: [email protected] (T.-t. Zhou), [email protected] (J.-r. Wu), [email protected] (Z.-y. Chen), [email protected] (Z.-x. Liu), [email protected] (B. Miao). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.brainres.2014.04.025 0006-8993/& 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Zhou, T.-t., et al., Effects of dexmedetomidine on P2X4Rs, p38-MAPK and BDNF in spinal microglia in rats with spared nerve injury. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.04.025

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1.

Introduction

Neuropathic pain, generally defined as a chronic pain state resulting from peripheral or central nerve injury, is characterised by symptoms such as allodynia, hyperalgesia and spontaneous pain (Woolf and Mannion, 1999). A growing body of evidence from diverse animal models of neuropathic pain showed that injury to the peripheral nervous system could lead to activation of microglia in the spinal cord and the consequent neuropathic pain, in which the activated microglia has an important role to play (Cao and Zhang, 2008; Milligan and Watkins, 2009; Tsuda et al., 2005; Watkins et al., 2001). Subsequent to peripheral nerve injury, the resting microglia in the spinal dorsal horn is converted to an activated state through a series of cellular and molecular modifications. Numerous studies have demonstrated that hypersensitivity to neuropathic pain is involved in P2 purinergic receptors (Burnstock, 2006a; Di Virgilio, 2006), especially P2X4R, which is a subtype of ionotropic purinoceptor. Spinal nerve injury can upregulate the expression of the P2X4Rs in the microglia in the spinal dorsal horn ipsilateral to the nerve injury, instead of the neurons or astrocytes wherein the upregulation of the P2X4Rs parallels the increase in pain hypersensitivity, while blockade of P2X4Rs can significantly attenuate allodynic effects (Tsuda et al., 2003, 2008 Ulmann et al., 2008). The activated P2X4Rs cause the phosphorylation of p38MAPK, resulting in the release of brain-derived neurotrophic factor (BDNF), all of which are essential to the persistence of pain hypersensitivity after nerve injury (Trang et al., 2009). It seems plausible that blockade of P2X4Rs-p-p38-MAPK-BDNF pathway in spinal cord may provide a novel therapeutic strategy for neuropathic pain. It is well established that α2-adrenoceptor agonists, like clonidine, have anti-nociceptive properties, and the pain modulatory action of the α2-adrenoceptor in the spinal cord has been most extensively studied (Duflo et al., 2002; Paqueron et al., 2003). Moreover, it has been found that the activation of α1-adrenergic receptor agonist interferes with α2-mediated analgesia (Gil et al., 2009). Therefore, it has been proposed that dexmedetomidine (DEX), a highly selective agonist of α2-adrenergic receptors clinically applied to sedation. Accumulating evidence has exhibited that DEX has a significant analgesic effect in many models of chronic pain (Guneli et al., 2007; Kimura et al., 2012; Liu et al., 2012). Despite the proposed mechanisms concerning DEX-induced analgesia, the possible interaction between DEX and P2X4Rs at a molecular level has not been elucidated. Our experiment was designed to investigate whether consecutive IP injections of DEX can attenuate neuropathic pain in rats with SNI by regulating the expressions of P2X4Rs, p-p38-MAPK and BDNF in spinal cord.

2.

Results

2.1. Decrease in the mechanical withdrawal threshold in SNI rats There were no significant differences in MWT between Normal and Sham groups (p40.05). In the SNI group, the MWT in the

hind limb ipsilateral to injury was significantly reduced as from postoperative day 1 until day 14 versus the Normal and Sham animals (po0.05) (Fig. 1).

2.2. Upregulation of P2X4R expression in hyperactive microglia in spinal cord in SNI rats Western blot showed apparently upregulated expression of P2X4Rs in the spinal dorsal horn in SNI rats versus the Normal and Sham groups. Immunofluorescence assay indicated that the average fluorescence intensities of P2X4Rs in the spinal dorsal horn ipsilateral to injury were significantly increased at days 7 and 14 after SNI versus the control groups (po0.05). Immunofluorescence assay results showed that the intensity of ionised calcium binding adapter molecule 1 (Iba1, a microglial marker) in the spinal dorsal horn ipsilateral to injury in SNI rats was increased, and the activated microglia exhibited enlargement of cell bodies with shrunk or thickened processes. One-way analysis of variance (ANOVA) showed that the average fluorescence intensities of Iba-1 in the spinal dorsal horn ipsilateral to injury began to increase on day 1 after SNI. Besides, on days 7 and 14, the average fluorescence intensities of Iba-1 were significantly greater in SNI rats versus the Normal and Sham groups (po0.05). On day 14, the average fluorescence intensities of P2X4Rs and Iba-1 were still greater, and exhibited yellow in colour, indicating that they were double-labelled. The results showed that P2X4Rs were mainly expressed in the microglia in the spinal dorsal horn ipsilateral to injury 14 days after sciatic nerve injury (Fig. 2A–D).

2.3. Time course of protein expression of p-p38 and BDNF in the spinal cord Western blotting showed that the protein expression levels of p-p38 and BDNF in the spinal dorsal horn in SNI rats were apparently upregulated versus the Normal and Sham groups

Fig. 1 – von Frey test for the assessment of mechanical paw withdrawal threshold (MWT) in the Normal, Sham and SNI groups. SNI-induced rats show a significant drop in the MWT from day 1 after operation, and maintain a lower level throughout the course of the experiment compared to Normal and Sham groups (n ¼8, npo0.05 versus Normal, # po0.05 versus Sham).



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Fig. 2 – Expression levels of P2X4Rs were increased, and exclusively co-localised in hyperactive microglia in the spinal cord. (A) Western blot showed an increase in the level of P2X4Rs protein in the spinal cord ipsilateral at different times after SNI. The time course of change in P2X4Rs protein were similar to that in MWT (n¼ 4, npo0.05 versus Normal, #po0.05 versus Sham). (B) Immunofluorescence revealed an extensive increase in P2X4R immunoreacivity in the ipsilateral lumbar spinal dorsal horn on day 7 and day 14. Scale bars¼ 100 μm. (n ¼4, npo0.05 versus Normal, #po0.05 versus Sham). (C) Immunofluorescence analysis with confocal microscopy indicated that an increase in Iba-1 (a microglial marker) immunoreactivity in the ipsilateral lumbar spinal dorsal horn on day 1, and an extensive increase on day 7 and day 14. Scale bars¼ 100 μm (n¼ 4, npo0.05 versus Normal, #po0.05 versus Sham). (D) Double immunofluorescence labelling of P2X4Rs with Iba-1. The result indicated that most P2X4R-positive cells were double-labelled (yellow) with Iba-1. This experiment was performed with spinal cord sections 14 days after nerve injury (n ¼4, Scale bars¼ 50 μm).

(po0.05), with no significant differences between the Normal and Sham groups (p40.05) (Fig. 3).

2.4. Increase in the mechanical withdrawal threshold in SNI rats by consecutive IP injections of DEX Compared with Shamþsaline and ShamþDEX groups, MWT in SNIþsaline group was significantly decreased (po0.05). Compared with SNIþsaline group, MWT was significantly increased in SNIþDEX group (po0.05), but still lower versus Shamþsaline and ShamþDEX groups. There were no significant differences in MWT between Shamþsaline and ShamþDEX groups (p40.05) (Fig. 4).

2.5. Effect of consecutive IP injections of DEX on the activation of microglia in the spinal dorsal horn ipsilateral to injury in SNI rats There were no significant differences in the population of Iba1-positive microglia between Shamþsaline and ShamþDEX groups on day 14 (p40.05). In comparison with Shamþsaline and ShamþDEX groups, the microglia in spinal dorsal horn ipsilateral to injury on day 14 after SNI were markedly hypertrophied, and the average fluorescence intensity of Iba-1 was significantly enhanced in the SNIþsaline group (po0.05). Compared with SNIþsaline group, after 14 consecutive days of IP injection of DEX 40 μg/kg, the average fluorescence intensity of microglia in spinal dorsal horn ipsilateral to injury was reduced (po0.05), but still greater


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versus the Shamþsaline and ShamþDEX groups (po0.05) (Fig. 5).

2.6. Effect of consecutive IP injections of DEX on the protein expressions of P2X4Rs, p-p38 and BDNF in the spinal dorsal horn ipsilateral to injury in SNI rats Western blot analysis revealed that IP injections of DEX for 14 consecutive days downregulated the protein expressions of P2X4Rs, p-p38 and BDNF in the spinal dorsal horn ipsilateral to injury versus SNIþsaline and ShamþDEX groups (po0.05), but still higher versus Shamþsaline and ShamþDEX groups, with no significant differences between the latter groups (p40.05) (Fig. 6).

3.

Fig. 3 – Marked upregulation of p-p38 and BDNF level in the spinal dorsal horn after SNI. Western blot showed an increase in the level of p-p38 and BDNF protein in the spinal cord ipsilateral at different times after SNI. The time course of change in p-p38 and BDNF protein are similar to that in MWT (n ¼4, npo0.05 versus Normal, #po0.05 versus Sham).

Fig. 4 – Increase in the mechanical threshold of SNI rats by intraperitoneal (IP) injections of dexmedetomidine (DEX, 40 μg/kg) for 14 consecutive days. von Frey test showed that IP injections of DEX 40 μg/kg for 14 consecutive days can increase the mechanical threshold of SNI rats compared to Shamþsaline and ShamþDEX groups, however, still lower than the controls (n ¼ 8, npo0.05 versus SNIþsaline, #po0.05 versus Shamþsaline, △po0.05 versus ShamþDEX).

Discussion

In the present study, the MWT in SNI rats was decreased as from postoperative day 1 and maintained at a low level throughout the experiment. The upregulation of P2X4Rs was detected as early as postoperative day 1. On day 14, the majority of P2X4Rs-positive cells were double-labelled with Iba-1, indicating the expression of P2X4Rs in microglia, which was consistent with previous reports (Tsuda et al., 2003). Time course of variations in P2X4Rs level in the spinal cord was in agreement with the decline of MWT. In addition, Western blotting showed that the protein expression levels of both p-p38 and BDNF were upregulated with the decline of MWT. IP administration of DEX 40 μg/kg for 14 consecutive days was effective against mechanical hyperalgesia in an SNI rat model of neuropathic pain. Western blot analysis revealed that consecutive IP injections of DEX downregulated the protein expressions of P2X4Rs, p-p38 and BDNF in the spinal dorsal horn ipsilateral to injury. Microglial cells, which account for 5–10% of the total cells in the central nervous system (CNS), are known as resident macrophages (Kreutzberg, 1996). Under normal conditions, microglial cells having a small soma with thin and branched processes are regarded as being quiescent. Despite the temporary quiescence, the microglial cells are in a de facto surveillance. In case of injury, their processes would rapidly migrate towards the site (Davalos et al., 2005; Nimmerjahn et al., 2005). Accumulating evidence from diverse animal models of neuropathic pain suggests that glia, particularly the microglial cells, play a critical role (Cao and Zhang, 2008; Milligan and Watkins, 2009; Tsuda et al., 2005; Watkins et al., 2001). In peripheral nerve injury, microglia in the spinal cord are activated. The activation of microglial cells involves both hypertrophy and hyperplasia in morphology, with the thickened and retracted processes (observed within the first 24 h after nerve injury) and increased cell population (observed around 2 to 3 days after nerve injury). These criteria are immunehistochemical markers for assessing the activation state of microglia in vivo (Raivich et al., 1999), wherein the variations of cell population is the most prominent event. Regardless of the various microglial populations in different models, peripheral nerve injury increases the number of dorsal horn microglia by 2 to 4 fold (Beggs and Salter, 2007). Activated microglia generate and release various algesic substances, such as pro-inflammatory cytokines, BDNF and other



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Fig. 5 – Effects of intraperitoneal (IP) injections of dexmedetomidine (DEX, 40 μg/kg) for 14 consecutive days on the activation of microglia in the ipsilateral of spinal dorsal horn of rats after SNI. Immunofluorescence analysis with confocal microscopy indicated that IP injections of DEX 40 μg/kg for 14 consecutive days inhibited microglial activation in the ipsilateral of spinal dorsal horn of rats after SNI (n ¼4, npo0.05 versus SNIþsaline, #po0.05 versus Shamþsaline, △p o0.05 versus ShamþDEX, scale bar¼100 μm).

mediators, all of which can enhance pain transmission (Watkins et al., 2001). Astrocytic activation generally follows that of microglial cells, but lasts longer and is involved in the persistence of pain (Hald et al., 2009; Mika et al., 2009; Trang et al., 2009). In contrast to the previous studies, our results showed that the microglial marker Iba-1 in the spinal dorsal horn in SNI rats was markedly upregulated on postoperative days 7 and 14, respectively. It is well known that activated microglial cells release many substances, which include BDNF, several types of cytokines and gaseous transmitter (De Leo et al., 2006; Kettenmann et al., 2011; Wieseler-Frank et al., 2004). Some of these substances were reported to rapidly modulate neuronal function by changing excitability and synaptic strength, leading to the central sensitization. The excitable neuronal, in turn, release much more ATP and excitatory amino acid (Kettenmann et al., 2013; Li et al., 2013; Liu et al., 2009). Such positive feedback loops facilitate persistent release of pain mediators and the consequent activation of microglia, leading to hyperalgesia and allodynia (Gwak et al., 2012; Watkins et al., 2001). These findings might account for the phenomenon that the microglia in our experiment still retained their activity on postoperative day 14. Purinergic receptors, which are potential regulators of microglial functions and can be activated by extracellular nucleotides, have been divided into two groups: P2X receptors (P2X1-7R) consisting of ligand-gated cation channel receptors,

and P2Y receptors (P2Y1, 2, 4, 6, 11-14R) consisting of metabotropic G-protein-coupled receptors (Burnstock, 2008; Burnstock et al., 2011; Inoue, 2002; Inoue, 2006; Khakh and North, 2006). Several purinergic receptors are expressed in microglial cells, including P2X4R, P2X7R, P2Y6R and P2Y12R (Boucsein et al., 2003; Kobayashi et al., 2008; Tsuda et al., 2003; Yiangou et al., 2006). Pharmacological, biochemical and molecular investigations have implicated P2X4Rs in microglial function. In our study, P2X4Rs were expressed in microglia. Pharmacological blockade of P2X4Rs reversed the established tactile allodynia, indicating that tonic activation of P2X4Rs in microglia is necessary for sustaining allodynia. Furthermore, mice lacking P2X4Rs fail to develop tactile allodynia after nerve injury. Moreover, spinal administration of P2X4Rs-stimulated microglia caused otherwise normal rats to develop allodynia. Therefore, P2X4Rs activation in microglia is not only necessary but also sufficient to cause tactile allodynia (Tsuda et al., 2003). BDNF, which is released from microglia, has been shown as a key protein mediating this microglia-neuron signalling (Coull et al., 2005): acting via its cognate receptor, tropomyosinrelated kinase B. BDNF signals to neurons in spinal lamina I to cause a rise in intracellular [Cl ], thereby counteracting gamma-aminobutyric acid- and glycine-mediated inhibitions in these cells. The disinhibition unmasks innocuous inputs to lamina I neurons and facilitates their responses to noxious


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Fig. 6 – Effect of intraperitoneal (IP) injection of dexmedetomidine (DEX, 40 μg/kg) for 14 consecutive days on the expressions of P2X4Rs, p-p38 and BDNF in the ipsilateral spinal dorsal horn of rats after SNI. Western blot showed that IP injections of DEX for 14 consecutive days, beginning at the time of SNI, decreased the expression of P2X4Rs; decreased microglial p38-MAPK phosphorylation, inhibited the expression of BDNF (n¼ 4, npo0.05 versus SNIþsaline, #po0.05 versus Shamþsaline, △p o0.05 versus ShamþDEX).

inputs (Keller et al., 2007). The resultant aberrant output in this normally nociceptive ascending pathway may serve as a basis for symptoms of neuropathic pain. Consistent with a role of BDNF as a key mediator of neuropathic pain, BDNF-/- mice display attenuated pain hypersensitivity after nerve injury compared with wild-type mice (Yajima et al., 2005). Moreover, in P2X4Rs null-mutant mice, BDNF accumulates in dorsal horn microglia after nerve injury, indicating that P2X4Rs are critical to the release of BDNF from microglia in vivo (Ulmann et al., 2008). Both the release and expression of BDNF are Ca2þdependent and are mediated via the phosphorylation of p38MAPK, a kinase implicated in pain hypersensitivity after peripheral nerve injury (Jin et al., 2003; Tsuda et al., 2004; Zhuang et al., 2007). P2X4Rs expressed on microglial cells are susceptible to ATP levels. Activation of these receptors instigates a series of conformational changes that allow entry of cations such as Ca2þ and Naþ into the cell via a non-selective channel (Burnstock, 2006b). Ca2þ that fluxes through the P2X4Rs is a critical intracellular step linking stimulation of these receptors to the phosphorylation of p38-MAPK. The phosphorylation of p38-MAPK by ATP is necessary for the release and expression of BDNF. Thus, these results indicate that P2X4Rs /p-p38/BDNF pathway plays an important role in neuropathic pain. In our present study, the expressions of P2X4Rs, p-p38 and BDNF were all upregulated in SNI rats. Dexmedetomidine (DEX) is a highly selective α2 adrenergic agonist with sedative properties. Despite its similarity to clonidine in mechanism of action, DEX has approximately an

eightfold α2-selectivity (Haselman, 2008). Unlike midazolam and propofol, DEX produces clinical sedation and analgesia without causing respiratory depression (Gerlach and Dasta, 2007). Moreover, intrathecal or systemic administration of DEX has analgesic effect in neuropathic pain models (Guneli et al., 2007; Kimura et al., 2012; Liu et al., 2012). There is accumulating evidence supporting the role of DEX in the inhibition of the microglia activation (Bell et al., 2014; Xu et al., 2010b). Meanwhile, DEX is a highly selective α2 adrenergic agonist, and therefore, doubts remain as to whether its inhibition of the microglia activation is mediated via the α2-adrenoceptor on the spinal microglia. Double immunofluorescence reportedly shows that α2-adrenoceptor are colocalised with NeuN (a neuron marker) and glial fibrillary acidic protein (GFAP; an astrocytic marker), but not with CD11b/c equivalent antibody (OX-42; a microglial marker) in the spinal dorsal horn (Xu et al., 2010a), suggesting the indirect effect of DEX on microglia. Despite the confirmed mechanisms underlying DEX-induced analgesia, the possible interaction between DEX and P2X4Rs in microglia at a molecular level has not been elucidated. As DEX is highly lipid-soluble and readily penetrates the blood–brain barrier following systemic administration, its IP injection was designated in this study. It was observed that IP injections of DEX for 14 consecutive days significantly reversed the mechanical hyperalgesia in SNI rats. The activation of microglia was markedly inhibited at the end of 14 consecutive days of DEX injection. The behavioural changes in the SNI rats were echoed by the markedly downregulated expressions of P2X4Rs, p-p38 and BDNF.



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Thus, one major mechanism by which DEX attenuates SNI-induced mechanical hyperalgesia might rest on the excitation of α2-adrenoceptor on the presynaptic membrane of neurons, which inhibit the release of norepinephrine and the transitive inhibition of Ca2þ influx, hence the reduced release of the algesic factors, such as ATP, neuropeptides and excitatory amino acids (Li and Eisenach, 2001). The inhibited activation of spinal microglia caused a decrease in the flux of Ca2þ, resulting in the inhibition of p38 phosphorylation, which in turn reduced the synthesis and release of BDNF, with the mechanical hyperalgesia significantly attenuated. In summary, in the present study, we examined the protective effect of DEX on SNI-induced neuropathic pain. The data suggested that the downregulation of P2X4Rs expressions, inhibition of p-38 phosphorylation and reduction of BDNF synthesis and release might account for the analgesic mechanisms.

4.

Experimental procedures

4.1.

Animals

Adult male Sprague-Dawley (SD) rats (Experimental Animal Centre, Xuzhou Medical College, Xuzhou, China) weighing 180–220 g were housed under controlled conditions of temperature (2172 1C), relative humidity (50%710%), and artificial lighting (lights on from 8 a.m. until 8 p.m.), with ad libitum access to food and water.

4.2.

SNI model

SNI model was induced with the method of Decosterd and Woolf (2000). Under pentobarbital anaesthesia (50 mg/kg, IP), the skin on the lateral surface of the thigh was incised and an incision was made directly through the biceps femoris muscle, with the sciatic nerve and its three terminal branches exposed: the sural, common peroneal and tibial nerves (Fig. 7). The SNI procedures comprised an axotomy and ligation of the tibial and common peroneal nerves leaving the sural nerve intact. The common peroneal and the tibial nerves were tight-ligated with 5.0 silk suture and sectioned distal to the ligation, with 274 mm of the distal nerve stump retained. Muscle and skin were wrapped in two layers. The rats in the sham-operated group underwent the same procedures except nerve ligation. Prior to experimental manipulation, the animals were acclimated for at least 5 consecutive days. All experimental protocols and animal handling procedures were approved by the Animal Care and Use Committee of Xuzhou Medical College and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimise the number of animal subjects and their suffering.

4.3.

Fig. 7 – Spared nerve injury (SNI) consists of ligation and transaction of the peroneal and the tibial nerves, leaving the third peripheral branch of the sciatic nerve, the sural nerve intact (Scholz et al., 2008).

4 rats were randomly sacrificed, with the L4-6 spinal cords isolated for Western blotting, while the other 4 rats were sacrificed, followed by the removal of L4-6 spinal cords for immunofluorescence assay. Part2: This experiment addressed the role of DEX on the neuropathic pain. Rats received IP injection of DEX (40 μg/kg) or equal dose of saline daily as from the end of the operation to day 14. Behavioural assessment was performed on 1, 1, 3, 5, 7, 14 days after SNI surgery. Rats were randomised into 4 groups (n¼8): shamþsaline, shamþDEX (40 μg/kg), SNIþsaline and SNI þDEX (40 μg/kg). On day 14 after behavioural evaluation, the rats were sacrificed, with the L4-6 spinal cord isolated for either Western blot or immunofluorescence analysis (n¼ 4 each).

4.4.

Reagents

The reagents included: DEX (Jiangsu Hengrui Medicine Co., Ltd., China); goat polyclonal anti-Iba-1 (Abcam, USA); rabbit polyclonal anti-P2X4Rs (Alomone Labs, Israel); Alexa 488 donkey anti-rabbit IgG, Alexa 594 donkey anti-goat IgG (Invitrogen, Carlsbad, CA, USA); mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-p-p38 (Cell Signaling Technology, USA); rabbit anti-BDNF (Sigma-Aldrich, St. Louis, MO, USA); horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Beyotime, China); enhanced chemiluminescence (ECL) (Beyotime, China).

Experimental groups

Part1: The rats were randomised into 3 groups (n ¼8): normal group (Normal group), sham-operated group (Sham group) and spared nerve injury group (SNI group). Animal behaviour was tested on 1, 1, 3, 5, 7, 14 days after SNI surgery. On days 1, 3, 5, 7, 14 after SNI surgery, after behavioural assessments,

4.5. von Frey test for mechanical paw withdrawal threshold The mechanical paw withdrawal threshold (MWT) was recorded before surgery (baseline, day 1) and postoperative days 1, 3, 5, 7 and 14 (Fig. 8). After a 30-min accommodation


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period, MWT was measured as the hind paw withdrawal responded to von Frey hair stimulation according to the updown method. All the behavioural tests were performed between 9 a.m. and 2 p.m. by an examiner blinded to the treatment groups. An ascending series of von Frey hairs with logarithmically incremental stiffness (0.40, 0.60, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, and 15.0 g) (Stoelting Co., Wood Dale, Illinois)was held in place for approximately 6–8 s, and each stimulus was applied 5 times at a 30-s interval. The trial began with the application of the 2.0 g von Frey hair. A positive response was defined as a withdrawal of the hind paw up on stimulus presentation. With a positive response to the stimulus, the next lower von Frey hair was applied, and in the case of a negative response, the next higher hair was applied. The 50% withdrawal threshold was determined using the up-down method of Chaplan and co-workers.

4.6.

Immunofluorescence measurement

Four animals in each group were randomly selected to be anesthetised after behavioural testing. The rats were intracardially perfused with saline and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), respectively. After the perfusion, the L4-6 segments of the spinal cord were removed, post-fixed in the same fixative for 12 h at 4 1C, and immersed in 30% sucrose in phosphate buffer at 4 1C for cryoprotection. Transverse spinal sections (30 μm) were cut in a cryostat and processed for immunofluorescence. All of the sections were blocked with 10% donkey serum in 0.01 M PBS (pH 7.4), with 0.3% Triton X-100 for 2 h at room temperature (r/t) and incubated over night at 4 1C with rabbit anti-P2X4Rs (1:100). The sections were incubated in Alexa 488 donkey anti-rabbit IgG or/and Alexa 594 donkey anti-goat IgG (1:200) for 2 h at

4 1C and thereafter rinsed in PBS. The omission of the primary antibody served as a negative control. All sections were cover-slipped with a mixture of 90% glycerin in 0.01 M PBS, followed by confocal microscopy (FV1000, Olympus Co. Ltd., Tokyo, Japan). The relative density of images was determined by subtraction from the background density in each image, with the mean density calculated.

4.7.

Western blotting analysis

At the end of the MWT measurements, the remaining four animals in each group were sacrificed. The L4-6 spinal cords from different groups were rapidly isolated. After dissection, all tissues were rapidly frozen in liquid nitrogen or stored at 80 1C for subsequent procedures. The frozen spinal cords were directly homogenised in a lysis buffer containing a cocktail of protease inhibitors. At the end of 15-min centrifugation at 12,000 rpm at 4 1C, the supernatant was collected for Western blotting. Equal amounts of protein (30 μg) were loaded in each lane and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 3% bovine serum albumin (BSA) for 2 h (r/t) and then rinsed thrice in Washing Buffer for 5 min each. Then they were sequentially incubated overnight at 4 1C with rabbit anti-P2X4Rs (1:500), rabbit anti-BDNF (1:1000), rabbit anti-p-p38 (1:1000), mouse anti-β-actin (1:1000) primary antibody. The blots were rinsed thrice in Washing Buffer for 5 min each and incubated for 1 h (r/t) with HRP-conjugated goat anti-rabbit IgG (1:1500) or goat anti-mouse secondary antibody IgG (1:1500). The blots were rinsed in Washing Buffer 4 times for 8 min, followed by visualisation using ECL and exposure onto X-films for 1–10 min. All western blot analyses were performed at least four times, with parallel results obtained. Squares of the same size from each band were measured for density, with the background subtracted. The expression of β-actin was used as a loading control for protein expression. The expression level of the proteins is an average of the densities per band area from each group.

4.8.

Statistical analysis

The data are expressed as the mean7SEM. Time courses measured for MWT were analysed by two-way ANOVA, followed by the Newman–Keuls post hoc test. Western blot and immunofluorescence analysis were performed by oneway ANOVA. po0.05 was considered statistically significant. All of the tests (including behaviour, Western blotting and immunofluorescence) were performed blindly. Fig. 8 – von Frey test for the assessment of mechanical paw withdrawal threshold (MWT) in the Normal, Sham, SNIþDEX 10 μg/kg, SNIþDEX 20 μg/kg, SNIþDEX 40 μg/kg groups. Consecutive IP injection of DEX 10 μg/kg or 20 μg/kg for 14 days did not reduce the MWT in the SNI rats. However, consecutive IP injection of DEX 40 μg/kg did attenuate the MWT, however, still lower than the controls (n ¼8, #po0.05 versus Normal, △po0.05 versus Sham, npo0.05 versus SNIþsaline).

Author contributions T.T.Z. participated in study design, experimentation, manuscript drafting and revision; J.R.W. participated in the design of the study and provided a critical revision of the paper; Z.Y. C. and Z.X.L. participated in the experimentation and the analysis of results; and B.M. was responsible for study design



and supervision, as well as data analysis and interpretation, manuscript drafting, revision and final approval.

Acknowledgments The present work was supported by grants from National Natural Science of China (81200861), the Jiangsu Provincial “Qinglan Project” Financing (53041212), and Xuzhou Science and Technology Plan Projects (XM12B017).

r e f e r e n c e s

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The antiallodynic action of pregabalin may depend on the suppression of spinal neuronal hyperexcitability in rats with spared nerve injury.

Elevated Neurosteroids in the Lateral Thalamus Relieve Neuropathic Pain in Rats with Spared Nerve Injury.

Effects of Dexmedetomidine combined with Dezocine on cognition function and hippocampal microglia activation of rats.

Analgesic effects and possible mechanisms of iridoid glycosides from Lamiophlomis rotata (Benth.) Kudo in rats with spared nerve injury.

[Effects of dexmedetomidine in conjunction with remote ischemic preconditioning on renal ischemia-reperfusion injury in rats].

The synergistic antinociceptive interactions of morphine and dexmedetomidine in rats with nerve-ligation injury.

Spared Nerve Injury Increases the Expression of Microglia M1 Markers in the Prefrontal Cortex of Rats and Provokes Depression-Like Behaviors.

Effects of calcitriol on experimental spinal cord injury in rats.

Dexmedetomidine attenuates blood-spinal cord barrier disruption induced by spinal cord ischemia reperfusion injury in rats.

Involvement of spinal microglia and interleukin-18 in the anti-nociceptive effect of dexmedetomidine in rats subjected to CCI.

The effects of dexmedetomidine on ischemia reperfusion injury in patients undergoing arthroscopy under spinal anesthesia.

Effects of tetramethylpyrazine on microglia activation in spinal cord compression injury of mice.

Upregulation of Chemokine CXCL12 in the Dorsal Root Ganglia and Spinal Cord Contributes to the Development and Maintenance of Neuropathic Pain Following Spared Nerve Injury in Rats.

Effects of dexmedetomidine on the protection of hyperoxia-induced lung injury in newborn rats.

reperfusion injury in rats.

Effects of dexmedetomidine on TNF-α and interleukin-2 in serum of rats with severe craniocerebral injury.

P2Y12 receptor-mediated activation of spinal microglia and p38MAPK pathway contribute to cancer-induced bone pain.

Arachidonic acid containing phosphatidylcholine increases due to microglial activation in ipsilateral spinal dorsal horn following spared sciatic nerve injury.

Polyamidoamine dendrimer-conjugated triamcinolone acetonide attenuates nerve injury-induced spinal cord microglia activation and mechanical allodynia.

The mGluR5 antagonist fenobam induces analgesic conditioned place preference in mice with spared nerve injury.

Differences in the phagocytic response of microglia and peripheral macrophages after spinal cord injury and its effects on cell death.

Effects of pulsed electromagnetic field and swimming exercise on rats with experimental sciatic nerve injury.

Accessory spinal nerve injury.

Mechanical sensitization of cutaneous sensory fibers in the spared nerve injury mouse model.