Exp Neurobiol 2018; 27(4): 267-276
Published online August 30, 2018
© The Korean Society for Brain and Neural Sciences
Jia-Piao Lin1,†, Chao-Qin Chen1,†, Ling-Er Huang1, Na-Na Li1, Yan Yang2, Sheng-Mei Zhu1, and Yong-Xing Yao1*
1Department of Anesthesia, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, P.R. China.
2Centre for Neuroscience, Zhejiang University School of Medicine, Hangzhou 310016, P.R. China.
Correspondence to: *To whom correspondence should be addressed.
TEL: 86-136-5666-8373, FAX: 86-571-8723-6169
†These authors are contributed equally
α2-Adrenoceptor agonists attenuate hypersensitivity under neuropathic conditions. However, the mechanisms underlying this attenuation remain largely unknown. In the present study, we explored the potential roles of purinergic receptor 7 (P2X7R)/extracellular signal-regulated kinase (ERK) signaling in the anti-nociceptive effect of dexmedetomidine in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve. An animal model of CCI was adopted to mimic the clinical neuropathic pain state. Behavioral hypersensitivity to mechanical and thermal stimuli was determined by von Frey filament and Hargreaves' tests, and the spinal P2X7R expression level and ERK phosphorylation were analyzed using western blot analysis and immunohistochemistry. In parallel with the development of mechanical and thermal hyperalgesia, a significant increase in P2X7R expression was noted in the ipsilateral spinal cord on day 7 after CCI. Intrathecal administration of dexmedetomidine (2.5 µg) for 3 days not only attenuated neuropathic pain but also inhibited the CCI-induced P2X7R upregulation and ERK phosphorylation. Intrathecal dexmedetomidine administration did not produce obvious effects on locomotor function. The present study demonstrated that dexmedetomidine attenuates the neuropathic pain induced by CCI of the sciatic nerve in rats by inhibiting spinal P2X7R expression and ERK phosphorylation, indicating the potential therapeutic implications of dexmedetomidine administration for the treatment of neuropathic pain.
Neuropathic pain, which affects 7%~8% of the population, is defined as a pathological pain caused by nerve lesions or diseases and is characterized by spontaneous pain, exaggerated responsiveness to noxious stimuli (hyperalgesia), and pain in response to normally non-noxious stimuli (allodynia) [1,2,3]. Conventional analgesics and treatments cannot satisfactorily alleviate neuropathic pain because of its unknown underlying pathogenesis and mechanisms, and therefore, neuropathic pain remains a vital major clinical and social problem .
The α2-adrenoceptor plays an important role in antinociception, and α2-adrenoceptor agonists attenuate hypersensitivity under neuropathic conditions [5,6,7]. There are three highly homologous subtypes of the α2-adrenoceptor: α2A, α2B, and α2C. Dexmedetomidine, a highly selective α2-adrenoceptor agonist, has been used for conscious sedation and as an adjunct for clinical anesthesia [8,9,10]. In animal experiments, dexmedetomidine has been shown to produce analgesic effects in models of neuropathic pain, but the mechanisms underlying these effects remain largely unknown [11,12,13].
Emerging evidence indicates that spinal microglia is activated after peripheral nerve injury and contribute to the development and maintenance of neuropathic pain . Previous studies have indicated that the purinergic receptor 7 (P2X7R), a subtype of the purinergic receptor predominantly expressed on microglia, is upregulated in the spinal cord after nerve injury . In addition, P2X7R seems to play an important role in neuropathic pain [16,17]. For example, P2X7R-knockout mice fail to develop behavioral hypersensitivity after partial sciatic nerve ligation . Moreover, P2X7 receptors are involved in regulating microglial activation via the release of cytokines . Recently, spinal microglia were shown to be involved in the anti-nociceptive effect of dexmedetomidine in rats subjected to chronic constriction injury of the sciatic nerve (CCI) . However, no study has examined the effects of dexmedetomidine on P2X7R activation and the related analgesic effects.
Extracellular signal-regulated kinases (ERKs, including ERK1 and ERK2), which are widely expressed in the neuronal cells, play important roles in central plasticity and modulation of pain processing [20,21]. In the spinal dorsal horn, ERKs are activated by innocuous stimuli in peripheral nerve-injured animals . Furthermore, the inhibition of ERK activation has been shown to reduce pain hypersensitivity and suppress brain-derived neurotrophic factor (BDNF) expression in the spinal cord [23,24], while BDNF is well known as an important factor for long-term memory . Recent studies have suggested that the ERK signaling pathway is involved in dexmedetomidine analgesia . However, the precise mechanism underlying the anti-nociceptive effects of dexmedetomidine in neuropathic pain remains unknown.
In the present study, we aimed to explore the possible roles of P2X7R in the anti-nociceptive effect of dexmedetomidine in a rat model of neuropathic pain following CCI. The results revealed the underlying mechanism of dexmedetomidine analgesia, indicating the therapeutic implications of dexmedetomidine administration for the treatment of neuropathic pain.
Male Wistar rats (weighing ~200 g on arrival) were obtained from the animal center of the Chinese Academy of Sciences and housed two per cage with water and food available
The rats were implanted with an intrathecal catheter under intraperitoneal anesthesia (pentobarbital, 60 mg/kg). A polyethylene-10 (Smiths Medical, UK) catheter was inserted into the subarachnoid space at the level of the spinal cord lumbar enlargement according to the method described by Størkson et al. . The animals were allowed to recover for 3 days before being randomly divided into groups. Rats showing neurological deficits postoperatively were discarded from the study.
Animals were randomly divided into naïve, sham, and CCI groups (n=6). To create a neuropathic pain state, we performed a surgical procedure according to the description reported by Bennett and Xie  and our recent study . Briefly, under pentobarbital (60 mg/kg, ip) anesthesia, the left sciatic nerve was exposed and freed from the underlying connective tissues. Three ligations were placed around the nerve with 4-0 chromic gut sutures, and a typical twitch of the hind paw was seen when the nerve was constricted. The overlying muscle and skin were closed with the same silk sutures. An identical surgical procedure was performed on sham-operated animals without ligation of the sciatic nerve. All animals received antibiotics (0.5 ml penicillin, 96 mg/ml, hypodermic injection) to reduce the possibility of infection. To reduce variability, all animal model procedures were carried out by one investigator who was proficient in modeling.
Animals were divided into naïve, sham, CCI+normal saline (NS), and CCI+DEXmedetomidine (DEX) groups (n=8 for each group). Dexmedetomidine (Sigma, Missouri) was diluted in NS. After neuropathic pain was successfully established, a dose of 2.5 µg DEX (in 20 µl volume) or NS (20 µl) was intrathecally administered once daily for three consecutive days from day 8 to 10 after CCI, followed by an injection of 10 µl NS to flush the catheter.
To minimize the stress induced by handling and measurements, we exposed the rats to the test condition for 3 days before the experiment. The behavioral test was performed 1 h after drug administration.
Western blot analysis was conducted as described in our previous reports . Rats were sacrificed immediately under deep anesthesia, followed by isolation of the lumbar 4~5 spinal cords. The spinal cord was divided into ipsilateral and contralateral halves, and then homogenized in ice-cold homogenization buffer (Beyotime Institute of Biotechnology, China). The homogenates were centrifuged at 10,000 ×
Immunohistochemical staining was conducted in accordance with our previous reports . Seven days after CCI or sham operation, rats were anesthetized and perfused thoroughly with NS, followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. Lumbar 4~5 segments were then harvested, postfixed, and dehydrated in 30% sucrose. Subsequently, 30-µm free-floating transverse sections were cut in a freezing microtome. Sections were collected and incubated for 48 h at 4℃ in rabbit anti-P2X7R (1:400; ProteinTech, IL) and rabbit anti-PERK (1:200; Millipore). The sections were then incubated with Cy3-conjugated anti-rabbit IgG (1:200; ProteinTech) for 2 h at room temperature. Positive signals were viewed with a fluorescence microscope (x-Cite 120; OLYMPUS, Japan) with the appropriate filters.
Data are expressed as the mean±S.E.M and were analyzed using one-way or two-way ANOVA followed by Fisher's least significant differences (LSD) test or Dunnett's multiple comparisons as appropriate. p<0.05 was considered statistically significant.
In the present study, a model of CCI was used to simulate a chronic neuropathic pain state. Using behavioral measurements, we found that the paw withdrawal thresholds for both mechanical and thermal stimuli were lower in the CCI group than in the sham-operated group on day 7, the time point at which the CCI group usually reached the maximal behavioral hyperalgesia after nerve injury . As shown in Fig. 1, compared with the sham group, the CCI group exhibited a significantly lower MT on day 7 after nerve injury (n=6, p<0.001; Fig. 1A), with similar results for the TL values (n=6, p<0.01; Fig. 1B), suggesting the development of robust mechanical and thermal hyperalgesia in the CCI rats. We further analyzed whether CCI-induced hyperalgesia is associated with spinal P2X7R activation. On day 7 after CCI, western blot analyses revealed that, in parallel with the behavioral hyperalgesia, P2X7R expression in the ipsilateral spinal cord was significantly higher than that in the sham group (n=4, p<0.05; Fig. 1D), while the expression in the contralateral spinal dorsal horn was not significantly different from that in the sham group (n=4, p=0.131; Fig. 1D). Similarly, 7 days after CCI, PERK expression in the ipsilateral spinal dorsal horn was significantly greater than that in the sham group (n=4, p<0.05; Fig. 1E), while the expression in the contralateral spinal dorsal horn was not significantly different from that in the sham group (n=4, p=0.234; Fig. 1E). The results of immunohistochemical assessments confirmed the activation of spinal P2X7R/ERK signaling after CCI injury (Fig. 1C).
To investigate the effect of α2-adrenoceptor agonists on CCI-associated neuropathic pain, which was reflected by mechanical and thermal hyperalgesia, we administered intrathecal dexmedetomidine (2.5 µg/20 µl) daily from day 8 to day 10 after CCI, when the pain behaviors were well established. Basal MT and TL were not significantly different among the groups before CCI surgery; however, the MT in the CCI+DEX group was significantly higher than that in the NS control (CCI+NS) groups from day 8 to day 10 after CCI (n=8, p<0.001; Fig. 2A), but it was still lower than that in the sham group (n=8, p<0.001; Fig. 2A). In contrast, the TL in the CCI+DEX group was also obviously higher than that in the CCI+NS group from day 8 to day 10 after CCI (n=8, p<0.001; Fig. 2B) and similar to that in the sham group, except on day 10 (p>0.05, from day 8 to 9; p<0.05, day 10, n=8; Fig. 2B).
Importantly, CCI surgery and dexmedetomidine administration did not produce significant effects on locomotor function. As shown in Fig. 3, CCI surgery and intrathecal dexmedetomidine administration at the dosage used in our study did not produce obvious effects on locomotor function. Furthermore, convulsions and hypermobility were not observed in any of the groups.
Next, to further explore the involvement of spinal P2X7R and ERK activation in dexmedetomidine analgesia, we examined the expression of P2X7R and ERK and its phosphorylated form (PERK) by western blot analyses. We chose to perform western blot analysis on day 10 after CCI on the basis of the results of behavior tests. After three days of dexmedetomidine or NS administration, the rats subjected to CCI were sacrificed 1 h after drug administration on day 10 after CCI surgery. In parallel with the changes in mechanical and thermal hyperalgesia, western blot analysis revealed that P2X7R expression in the CCI+NS group was highly upregulated compared with the expression in the sham group (n=5, p<0.01; Fig. 4A). However, in contrast to the CCI+NS group, the CCI+DEX group exhibited significant downregulation of P2X7R expression (n=5, p<0.01; Fig. 4A). Furthermore, western blot analysis showed that although there was no obvious difference in the total ERK expression in the spinal cord on day 10 following CCI among the naïve group, CCI+NS group, or CCI+DEX group (n=4, p>0.05; Fig. 4B), PERK expression in the CCI+NS group was significantly upregulated in comparison with that in the sham group (n=5, p<0.05; Fig. 4C); however, in parallel with the changes in mechanical and thermal hyperalgesia, the expression of PERK in the CCI+DEX group was significantly lower than that in the CCI+NS group (n=5, p<0.05; Fig. 4C).
Similar to previous reports, we found that CCI rats showed robust behavioral hypersensitivity to mechanical and thermal stimuli 7 days after nerve injury, and this hypersensitivity lasted beyond the entire experimental period. Using protein analysis, we also found that P2X7R expression was upregulated in the ipsilateral spinal cord in parallel with the CCI-induced behavioral signs of mechanical and thermal hyperalgesia, indicating that spinal P2X7R is involved in the development of neuropathic pain. The results are consistent with those from a previous study conducted by Kobayashi et al. , which showed that P2X7R protein expression increased in the spinal cord in a spared nerve injury model of neuropathic pain, peaking at 7 d after injury.
Dexmedetomidine, a selective α2-adrenoceptor agonist, has been used for conscious sedation and as an adjunct for clinical anesthesia in recent years [8,9,10]. In the present study, we found that intrathecal administration of dexmedetomidine attenuated the behavioral hyperalgesia induced by CCI in rats. In a previous study, Li et al.  found that intrathecal dexmedetomidine produced an anti-allodynia effect in a rat pain model. However, in the current study, we demonstrated that dexmedetomidine attenuated not only mechanical allodynia but also thermal hyperalgesia. In another report, Liu et al.  found that systemic administration of dexmedetomidine attenuated thermal and mechanical hyperalgesia in a rat model of partial sciatic nerve ligation (PSNL). Our study further confirmed the previous results that dexmedetomidine attenuates the neuropathic pain induced by nerve injury.
P2X7R, an ATP-gated nonselective cation channel, has been studied as a subtype of purinergic receptors. Spinal P2X7R has been reported to be involved in acute and chronic pain [37,39,40]. P2X7R blockade with an antagonist or genetic knockout of P2X7R reduced the mechanical and thermal hyperalgesia induced by nerve injury . He et al.  found that spinal P2X7R mediates microglial activation-induced neuropathic pain in a sciatic nerve injury rat model. All of these observations indicate that P2X7R plays a vital role in the development of neuropathic pain induced by multiple causes. Nevertheless, the specific mechanism by which spinal P2X7R mediates neuropathic pain is still largely unknown. Electrophysiological studies have revealed that stimulation of P2X7R leads to reversible Ca2+ and Na+ influx and associated membrane voltage changes . Ca2+ influx has been widely viewed as the pivotal stimulus to elicit cell reactions and activate intracellular signaling pathways [43,44], including ERK. As one of the classical mitogen-activated protein kinase (MAPK) signaling pathways, ERK may be activated by Ca2+, protein kinase C (PKC), and growth factors to regulate cell activities. The activated form of ERK, PERK, stimulates the production and release of inflammatory molecules, amplifies pain signals, and contributes to the development of neuropathic pain [45,46,47].
In the present study, we found that the expression of spinal P2X7R and the phosphorylated (and thus activated) forms of ERK were upregulated following CCI, although total ERK expression was not altered by CCI injury, suggesting that in the spinal cord, ERK contributes to pathological pain hypersensitivity via its phosphorylation, not its protein expression. This finding is in agreement with previous studies [48,49]. Moreover, the current study investigated the possible involvement of ERK phosphorylation in the process of dexmedetomidine analgesia. While dexmedetomidine attenuated behavioral hyperalgesia, it inhibited ERK phosphorylation as well, suggesting that spinal ERK is involved in dexmedetomidine analgesia. This finding was supported by studies showing that dexmedetomidine inhibited the intracellular ERK signaling pathway activated by nerve injury .
Importantly, we found for the first time that while dexmedetomidine attenuated behavioral hyperalgesia, it also downregulated P2X7R overexpression in the spinal cord after CCI. P2X7R activation leads to the activation of MAPK pathway proteins, which are known to participate in the development of chronic pain . A possible mechanism for the involvement of P2X7R and the ERK signaling cascade in neuropathic pain may be as follows. When activated, P2X7R initiates a positive feedback loop between ERK phosphorylation and tumor necrosis factor alpha (TNFα) production, thus inducing hyperalgesia and allodynia . In addition, P2X7R has been shown to activate ERK1 and ERK2 independently of Ca2+ influx . The exact mechanism that mediates dexmedetomidine analgesia is still unknown. However, previous studies have found that dexmedetomidine inhibits elevations in intracellular Ca2+ levels in cultured rat dorsal root ganglia cells by its α2-adrenoceptor agonist effect . Taken together, these observations suggest that dexmedetomidine may decrease postsynaptic intracellular Ca2+ concentrations by acting at presynaptic α2-adrenoceptors, thereby affecting the expression of P2X7R and ERK phosphorylation. Nevertheless, the precise mechanism by which dexmedetomidine mediates the expression of P2X7R and suppresses the phosphorylation of ERK needs to be further elucidated.
In conclusion, our study demonstrated that spinal dexmedetomidine attenuates the neuropathic pain induced by CCI of the sciatic nerve in rats, which might be attributable to its inhibition of P2X7R expression and ERK phosphorylation, suggesting the potential therapeutic implications of dexmedetomidine administration for the treatment of neuropathic pain.