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Local injection to sciatic nerve of dexmedetomidine reduces pain behaviors, SGCs activation, NGF expression and sympathetic sprouting in CCI rats
Accepted Manuscript Title: Local injection to sciatic nerve of DEX reduces pain behaviors, SGCs activation, NGF expression and sympathetic sprouting in CCI rats Authors: Jing-ru Wu, Hui Chen, Deng-xin Zhang, Kai Jiang, Ying-ying Yao, Ming-ming Zhang, Bo Zhou, Jie Wang PII: DOI: Reference:
S0361-9230(17)30121-1 http://dx.doi.org/doi:10.1016/j.brainresbull.2017.04.016 BRB 9210
To appear in:
Brain Research Bulletin
Received date: Accepted date:
1-3-2017 27-4-2017
Please cite this article as: Jing-ru Wu, Hui Chen, Deng-xin Zhang, Kai Jiang, Ying-ying Yao, Ming-ming Zhang, Bo Zhou, Jie Wang, Local injection to sciatic nerve of DEX reduces pain behaviors, SGCs activation, NGF expression and sympathetic sprouting in CCI rats, Brain Research Bulletinhttp://dx.doi.org/10.1016/j.brainresbull.2017.04.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Local injection to sciatic nerve of DEX reduces pain behaviors, SGCs activation, NGF expression and sympathetic sprouting in CCI rats Jing-ru Wua,1, Hui Chena,1, Deng-xin Zhangb, Kai Jiangc, Ying-ying Yaoa,, Ming-ming Zhang a,, Bo Zhoua, Jie Wanga,*
a Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu, China, 221002 b Department of Anesthesiology, The Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China, 214000 c Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China, 215000
*Corresponding author: Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu, China, 221002 Fax:+86 15895208059 E-mail addresses: [email protected] (J. Wang) 1
These two authors contributed equally to this work.
Other authors E-mail address: [email protected] (J.-r.Wu), [email protected] (H. Chen), [email protected](D.-x. Zhang), [email protected] (K. Jiang), [email protected] (Y.-y. Yao), [email protected](M.-m. Zhang), [email protected] (B.Zhou)
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Highlights We examined the anti-nociceptive effects of DEX on peripheral nervous system. Local injection to sciatic nerve with DEX can inhibited the heat hypersensitivity. Pre-treat by DEX inhibited the pathological changes in DRGs. Post-treat by DEX had no significant effect on the induced pathological changes.
Abstract Neuropathic pain has become an intractable health threat, with its profound effect on quality of life. Dorsal root ganglia (DRG) is evidenced to play a crucial role in neuropathic pain. The peripheral nociceptive afferents seem to be essential not only to initiate the process of neuropathic pain, but also to maintain and modulate it. Dexmedetomidine (DEX), a highly selective agonist of α2-adrenergic receptor (α2-AR), has provided significant analgesia in neuropathic pain. In the present study, we found that local injection to sciatic nerve of DEX alleviated heat hypersensitivity induced by chronic constriction injury (CCI). Western blotting revealed that DEX inhibited the overexpression of nerve growth factor (NGF) significantly. Immunohistofluorescence results showed that DEX inhibited glia cells activation and sympathetic sprouting simultaneously in DRG. Our study suggests that DEX attenuates neuropathic pain in CCI rats by down-regulation of satellite glial cell (SGC) activation, NGF expression and sympathetic sprouting.
Abbreviations: DRG, dorsal root ganglia; DEX, dexmedetomidine; α2-AR, α2-adrenergic receptor; CCI, chronic constriction injury; NGF, nerve growth factor; SGC, satellite glial cell; PWL , paw withdrawal latency; GFAP, glial fibrillary acidic protein. Keywords: Neuropathic pain; Dexmedetomidine; Satellite glial cell; NGF; sympathetic sprouting; microglia
1. Introduction Numerous studies have shown that peripheral nerve injury is often accompanied by neuropathic pain. The development and maintenance of neuropathic pain are dependent on nociceptive input. Dorsal root ganglia (DRG) is an important part of peripheral nervous system. Pathological changes in DRG are critical to neuropathic pain. DRG contains primary sensory neurons, which is the first stage of nociceptive information input. After peripheral nerve injury, primary sensory neurons receive peripheral afferent information and pass it to the spinal cord dorsal horn, completing the delivery of nociceptive information (Manzano et al.,2008). -2-
Satellite glial cell (SGC) is the main type glia cell in DRG. In respond to peripheral nerve injury, the resting SGCs converted to an activated state through a series of cellular and molecular modifications (Hanani, 2005). By releasing inflammatory cytokines, chemokines and other factors known to facilitate pain signalling, SGCs play important roles in mediating neuropathic pain. It was reported that there is an increase of nerve growth factor (NGF) in DRG following peripheral nerve injury. Further study showed NGF mRNA and protein levels within the SGCs are elevated (Sebert and Shooter,1993; Wells et al.,1994; Zhou et al.,1999). NGF is widely distributed in the central and peripheral nervous system. NGF is the member of the neurotrophin family, which is essential for the survival and growth of sympathetic and sensory neurons(Lindsay,1994; Patel,2000). Studies found that NGF was involved in the pathophysiology of pain in many neuropathic pain models (Choi and Son, 2011; Belrose et al., 2014; Richards and Mcmahon, 2013). Evidence implicate that NGF is a peripheral pain mediator by working as a sensitizer of nociceptors (Crowley et al.,1994; Frade and Barde,1998; Campenot,1987; Woolf et al.,1994). Some studies showed that NGF over-expression evoked nociceptive behavior in animals and humans (Lewin and Mendell,1996; Lewin and Mendell,1994; Pertens et al.,1999; Malin et al.,2006). Experimental blockade of NGF can decrease afferent nerve activity, nociceptor sensitivity, therefore preventing hyperalgesia (Djouhri et al.,2001; McMahon et al.,1995; Lewin and Mendell,1996). By affecting the release of inflammatory mediators, promoting the growth of nerve fibers and binding with tyrosine kinase(TrkA) receptor for regulating ion channels and molecular signals, NGF plays an important role in the pathophysiology of pain (Richardson and Mcmahonsb, 2013). Dexmedetomidine (DEX) is a highly selective α2-AR agonist which has been extensively employed in anaesthesia and critical care medicine for its anxiolytic, sedative and analgesic effects (Kamibayashi and Maze, 2000; Coursin, 2001; Paris and Tonner, 2005). Numerous studies have showed that DEX exhibits a significant analgesic effect in models of chronic pain (Guneli et al., 2007; Kimura et al., 2012; Liu et al., 2012). DEX stimulates α2-AR in the locus coeruleus and spinal cord providing sedation and analgesia (Sonohata et al., 2004). Our study was designed to investigate whether DEX has an effect on peripheral nervous system in model of CCI rats.
2. Experimental procedures 2.1 Animals Male Sprague-Dawley rats (200–250g) from the experimental animal center(Xuzhou Medical University, China), housed at a constant ambient temperature of 24 ± 1 ◦C under a 12 h light/dark cycle with ad libitum access to food and water.All experiments were conducted in accordance with the guidelines of the International Association for the Study of Pain and were approved by the Committee for the Ethical Use of Laboratory Animals, Xuzhou Medical University. 2.2 Experimental groups -3-
Part1: The rats were randomly divided into 2 groups (n=28): Sham group and CCI group. Animal behavior was tested on -1, 1, 3, 7, 14 days . Part2: This experiment addressed the role of DEX on the neuropathic pain. Rats received Local injection to sciatic nerve of DEX (25μg/kg) or equal dose of NS daily as from the end of the operation to day 6. Rats were randomly divided into 4 groups(n=12): Sham+NS, Sham+DEX, CCI7d+NS and CCI7d+DEX. Animal behavior was tested on -1, 3, 5,7 days after surgery. Part3:In this part, rats were randomly divided into 4 groups(n=12): Sham+NS, Sham+DEX, CCI14d+NS and CCI14d+DEX and received local injection to sciatic nerve of DEX (25μg/kg) or equal dose of NS daily as from day 7 to day 13. Animal behavior was tested on -1, 5, 7, 10, 12,14 days . 2.3 CCI and local injection to sciatic nerve model The CCI model was created as described by Bennett and Xie(Bennett and Xie,1988). Animals were anesthetized with 10 % chloral hydrate (300 mg/kg, i.p.), and the left sciatic nerve was exposed. Four loose ligatures were made around the nerve using 4–0 braided silk thread (Ethicon Inc., Brussels,Belgium) at intervals of 1 mm. The incision was closed in layers. In sham-operated rats, the same procedure was performed without sciatic nerve ligation. According to the method reported by Leszczynska and Kau(Leszczynska and Kau,1992),rats were anesthetized with Sevoflurane, drugs were injected into the area of the popliteal fossa of the left hind limb by hamilton syringe. The rats will be checked for motor impairments after drug injection. 2.4 Behavioral test Behavioral test was performed 1 hour later after drug injection. The behavioral testing was performed by observers who were blinded to the experimental conditions. To assess paw withdrawal latency(PWL), rats were placed on glass floor of a thermal apparatus (IITC Plantar Analgesia Meter,IITC Life Science Inc)30 min for accommodation before the PWL assessment. A light of variable intensity could be applied to the plantar hindpaws.Each rat was tested five times alternately, allowing sufficient time between tests to allow paw temperatures to return to baseline.25s was set as the cutoff time to avoid tissue damage. 2.5 Immunofluorescence Method After behavioural testing, the rats were subjected to deep anesthesia with 10 % chloral hydrate (400 mg/kg, i.p.) and transcardial perfusion with 0.01 MPBS (200 mL, pH 7.4) followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB; 300 mL,pH 7.4) for fixation. The L4-5 segments of the spinal cord and DRG were removed and post-fixed for 4–6 h at 4◦C, then immersed in 30% sucrose in phosphate buffer at 4 ◦C. The tissues were embedded in optimal cutting temperature compound at -20◦C and sectioned on a cryostat (Leica CM1950,Germany). Transverse spinal sections were cut at the thickness of 35μm and The ganglia were sectioned with a vibratome at a thickness of 30μm, along the long axis of the DRG, The sections were washed with 0.3 % Triton -4-
X-100 for 15min and incubated with donkey serum for 2 h at room temperature. Spinal slices were incubated with goat anti-Iba-1(1:300; Abcam, USA).Half of the ganglia slices were incubated with rabbit anti-NGF(1:200;Abcam,USA)and mouse antiGFAP(1:400;Cell Signaling Technology,USA),other slices were incubated with rabbit anti-NeuN(1:400;Cell Signaling Technology,USA)and mouse anti-Tyrosine Hydroxylase(1:300; Millipore, Bedford, MA, USA).All the slices were then incubated at 4°C for 24 hours. After three rinses in PBS-T, the spina sections were incubated for 2 hours at room temperature in Alexa Fluor 594 donkey anti-goat IgG(1:200; Invitrogen, Carlsbad, CA, USA),and the ganglia sections were incubated in Alexa 488 donkey antimouse IgG(1:200; Invitrogen, Carlsbad, CA, USA) and Alexa 594 donkey anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA, USA). Finally, after these slices rinsed, adhered onto the microslide. Each microslide was examined under the confocal laser microscope (FV1000; Olympus, Tokyo, Japan). 2.6 Western Blotting After the behavioral tests. L4-5 DRG of the rats were isolated and stored at -80 ◦C. The ganglias and 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 ◦C, the supernatant was collected for Western blotting. Equal amounts of protein (30 μg) were loaded in each lane and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Themembranes were then sealed with the addition of 5 % skim milk in Tris-buffered saline (TBS–Tween) for 1 h at room temperature, followed by incubation with rabbit anti-NGF(1:800; Abcam, USA) and rabbit anti-β-actin (1:1000; ZSGB-Bio, China) primary anti-body at 4 ◦C overnight. The membranes were rinsed in Washing Buffer for 3 × 10 min. This was followed by incubation for 2 h at room temperature with a peroxidase-conjugated goat anti-rabbit IgG antibody (1:1000; Beyotime, China). The membranes were washed 3 × 10 min in TBS-T. Immunoreactivities were visualized by NBT/BCIP assay kit (Sigma-Aldrich). Finally, Western blot densitometry analysis of signal intensity was performed using ImageJ software. 2.7 Statistical analysis The data were represented as mean ± SEM. Statistical analysis was performed using Prism 6.0(GraphPad) software. One-way ANOVA for data from different groups and two-way ANOVA with on repeated factor for data of PWL were employed. p < 0.05 was considered statistically significant. All of the tests including behavior, Western blot and immunoflurorescence were performed blinded.
3. Results 3.1 CCI induced changes of SGCs activation, NGF expression and sympathetic sprouting in DRG In DRG sections of CCI rats, glial fibrillary acidic protein (GFAP) -5-
immunofluorescence intensity in SGCs encircling the neurons was measured as indication of activation. The results showed that GFAP level increased as early as day 1 after CCI. The increase was significant on day 7 and 14, as compared to the Sham group (p<0.05; Fig.1A). The NGF level also increased after CCI, and is co-localized with GFAP activation in SGC (Fig.1B). Western blotting confirmed the increase of NGF was significant from day 3 to day 14 of CCI (p<0.05; Fig.1C). To quantify sympathetic sprouting after the nerve lesion, basket formations around some neurons was measured in fixed sections by immunostaining for tyrosine hydroxylase. The results showed increase of basket formations beginning on day 3 after CCI versus the Sham group (p<0.05; Fig.2A). 3.2 CCI induced microglia activation in the spinal dorsal horn In the spinal cord, the intensity of ionized calcium binding adapter molecule 1 (Iba-1, a microglia marker) was measured and normalized. The results indicated that the fluorescence intensity of Iba-1 in the ipsilateral spinal dorsal horn increased significantly after CCI surgery versus the Sham group (p<0.05; Fig.2B). 3.3 Effect of pre-treatment by local injection to sciatic nerve with DEX after CCI surgery Rats received local injection to sciatic nerve (DEX 25 μg/kg) or equal dose of NS daily after surgery until day 6. Behavioral test was performed 1 hour later after the injection.The results showed there were no significant differences in PWL between Sham+NS and Sham+DEX groups (p>0.05). Compared with the Sham groups, PWL in CCI7d+NS group was significantly decreased (p<0.05). The PWL of CCI7d+DEX group was significantly increased versus CCI7d+NS group, but still lower than the Sham groups (p<0.05)(Fig.3A). Intensity of GFAP and Iba-1, protein expression of NGF and basket formations in CCI7d+DEX group was significantly lower than those in the CCI+NS group (p<0.05), but still greater than those in the Sham groups (p<0.05). The results showed the injections from day 0 to day 6 significantly inhibited the glia activation,up-expression of NGF and sprouting of sympathetic fibers (Fig.3B-C;Fig.4A-B). 3.4 Effect of post-treatment by local injection to sciatic nerve with DEX after CCI surgery In this part, rats received local injection to sciatic nerve of DEX (25 μg/kg) or an equal dose of NS daily as from day7 to day13. Compared with Sham groups, the PWL in CCI14d+DEX group was significantly decreased from day10 to day14, but showed a significant increase than CCI14d+NS group (p<0.05; Fig.5A). Compared with CCI14d+NS group,the intensity of GFAP and Iba-1, protein expression of NGF and basket formations in CCI14d+DEX group had no significant differences (p>0.05; Fig.5B-C;Fig.6A-B).
4. Discussion In the group without DEX, the PWL in CCI rats decreased from as early as day1 and maintained at a low level throughout the experiment. We found CCI surgery alone induced SGCs activation, followed by NGF up-regulation and sympathetic sprouting at -6-
DRG. CCI surgery also induced microglia activation in the spinal dorsal horn. These results are consistent with previous studies. Pre-treat by local injection to sciatic nerve of DEX after CCI surgery for 7 consecutive days was effective in preventing heat hypersensitivity and related pathological changes. Post-treat by local injection to sciatic nerve of DEX can alleviate heat hypersensitivity, but had no significant effect on the induced pathological changes. SGCs are glial cells wrapped around the primary sensory neurons in the peripheral nervous system. Though much less abundant than astrocytes and microglia in the spinal cord, SGCs are the main type of glia cell in the DRG(Hanani, 2010). Due to its unique location in the sensory ganglia, SGCs are capable of exerting a strong influence on pain transmission. In respond to nerve injury, SGCs upregulate GFAP protein expression, undergo cell division and release various algesic substances (Elson et al., 2004b; Zhang et al., 2009). SGCs express a series of receptors which can respond to the neurotransmitters released by neurons. By participating in the exchange of information among neurons, these neurotransmitters play important roles in the control of neuronal microenvironment and mediation of neuronal excitability (Weick et al., 2003; Ceruti et al.,2008).Activated SGCs generate and release inflammatory cytokines and neurotransmitters such as bradykinin, interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α), neurotrophins, and ATP into its surroundings. SGC modulate pain by influencing neighboring neuron’s function and status with those released chemicals (Suadicani et al., 2010; Takeda et al., 2008; Zhang et al., 2007). NGF,which can be released from SGC is one of the most important molecular mediators of peripheral sensitization (Crowley et al., 1994; Frade and Barde, 1998; Campenot, 1987). NGF regulates expression of various pain-related proteins in nociceptive sensory neurons, including substance P, Navl.8 sodium channel, brainderived neurotrophic factor (BDNF), etc. These proteins further sensitize nociceptive neurons, promoting the activation of secondary neurons in the central nervous system (Mantyhpw et al., 2011；Balaratnasingam and Janca，2012；Schnegelsberg et al., 2010; Nishigami et al., 2013). NGF participates in mediating inflammatory and immune responses after peripheral nerve injury (Herzberg et al., 1997). By activating mast cells, neutrophils and eosinophils, releasing a variety of pain mediators such as 5-HT, prostaglandins, bradykinin, IL-1β, TNF-α, histamine and other mediators of inflammation, amplifying the inflammatory response, NGF stimulates the nociceptors and promotes the generation of pain (Schnegelsbergb et al.,2010;Nishigamit et al., 2013). NGF mediates ion channel opening such as those expressed on sensory neurons the sodium channel Nav1.8, which is involved in NGF-induced thermal hyperalgesia (Kerr, 2001). Evidence shows that NGF modulates this sodium channel current through the protein kinase A pathway following TrkA binding (Brackenbury, 2007). Since many NGF-responsive neurons contain the vanilloid receptor TRPV1, it is suspected that this receptor plays a role in NGF-mediated hypersensitivity (Ng et al., 2011). Stimulations cause TRPVl opening and Ca2+ influx, generating action potentials in nociceptive neurons for pain signal conduction (Ramsey et al., 2006). Studies have shown that NGF increases binding of NGF to TrkA receptors on nociceptive neurons activated -7-
phospholipase C and reduced TRPVl opening threshold, thus inducing neurons sensitivity. NGF increased TRPVl expression and promoted its insertion into the plasma membrane (Donnerer et al., 2005; Xue et al., 2007; Zhang et al., 2005). We speculate that by these NGF reduces the action potential (AP) threshold of nociceptive neurons. Under normal physiological conditions, the sympathetic nervous system is not directly associated with peripheral nerve sensory. NGF up-regulation is concomitant with an increase in overall sympathetic fiber density in the cellular region of the DRG, contributing to the appearance of “basket’’ formations (Chien et al., 2005; Pertin et al., 2007). NGF alters expression of ion channels and receptors, as the result of sensory neurons remodelling contributes to the hypersensitive to noradrenaline released by Sympathetic fibers(McLachlan et al., 1993; Xie et al., 2010; Zhang and Tan,2011). The abnormal sympathetic-sensory coupling and the sympathetic sprouting involved in the formation and modulation of neuropathic pain will cause paresthesia or hyperalgesia (Pertin et al., 2007; Xie et al., 2007). DRG is an important part of the coupling after peripheral nerve injury. Microglia activation is dependent on nociceptive input. After nerve injury, the sensory neurons activate microglia by releasing neurotransmitters and inflammatory mediators. Microglia activation mainly manifests as morphological changes: limb-like protrusion disappears and becomes amoeba-like, accompanied by specific cell marker expression and enhanced cell proliferation. By autocrine and paracrine, its activation is further strengthened(Grace et al., 2011). Our results showed that the microglial marker Iba-1 was significantly upregulated until to day 14 after CCI surgery. Activated microglia produces and releases large amounts of inflammatory cytokines, modulating neuronal excitability. Activated microglia can act on the surrounding non-activated astrocytic to induce inflammation and immune responses. These lead to hyperalgesia and allodynia(Griffin et al., 2007; Tsuda et al., 2005; Scholz and Woolf, 2007). In current study, DEX inhibits of the microglia activation. Like previous reports, nerve injury increases SGCs and microglia activation and that many pain treatments reduce such increases. This effect could result from the reduction of nociception input by the drug, not by the drug itself. Multiple studies have demonstrated that systemic or intrathecal administration of DEX produces significant anti-nociceptive effects in models of neuropathic pain.DEX produces its effect by stimulating α2-ARs in sensory neurons. Activation of this receptor leads to a series of effects, such as reduction of cyclic AMP, protein kinase A and Na+ channels activation. Attenuation of hyperalgesia by DEX is dependent on the activation of α2-ARs on the presynaptic membrane of neurons. DEX reduced the release of pronociceptive neurotransmitters, such as glutamate from the primary afferent terminals, hyperpolarized spinal interneurons, resulting in suppression of pain signals transmission (Poree et al., 1998). Study showed DEX affected the locus coeruleus and was dependent on the noradrenergic pathways. By inducing presynaptic membrane depolarization and inhibiting the release of substance P, NE, or other “harmful” peptides, DEX suppressed the transmission of nociceptive stimulation in the spinal dorsal horn (Nelson et al., 2003). In peripheral nervous system, DEX inhibited APs conduction by depressing voltage-gated Na+-channel currents (Oda et al., 2007). Furthermore, it has -8-
been reported that systemic administration of DEX, which suppresses the expression of mRNAs encoding inflammatory cytokines in inflammatory cells, inhibiting the amplification of inflammatory response (Kanazi et al., 2006). Some studies showed mix DEX with local anesthetics can enhance its effectiveness, shortening sensory and motor block onset and extended the analgesia duration(Memis ¸et al., 2004; Kanazi et al., 2006). Due to this, we chose DEX given perineurally in the current study. Pre-treat by local injection to sciatic nerve of DEX after nerve injury alleviated heat hypersensitivity significantly. In DRG,DEX inhibited SGCs activation, NGF over-expression and sympathetic sprouting. The above results suggest inhibition of spontaneous activity occurred with the use of DEX, as evidenced by the reduction of peripheral input through inhibition of microglia activation in spinal dorsal horn. However, local injection to sciatic nerve from day 7 after surgery showed little effect as compared with those which started DEX immediately after the nerve injury. This interesting result suggests that SGCs activation and subsequent pathological changes are irreversible, and treatment should follow the principle of preemptive analgesia, i.e. The treatment should begin at the initial stages of the pain. Once neuropathic pain has already been established, it is much more difficult to reverse the biological and biochemical changes in the activated cells. In the current study, the rats receiving DEX didn’t show motor impairments, as evidenced by little difference in heat hypersensitivity among Sham+NS and Sham+DEX groups. This shows that DEX does not produce a nerve blockade as local anesthetics. DEX produces its effect probably by stimulating α2-ARs in sensory neurons, reducing its reaction to damage, enhancing the ability of anti-nociception. DEX increases the AP threshold of nociceptive neurons, reducing the reaction to neurotransmitters and inflammatory cytokines. Stimulating α2-ARs can inhibit the upregulation of some receptors and sodium channels in sensory neurons. DEX may also inhibit the generation and release of inflammatory cytokines and chemokines by SGCs. In summary, in the present study, we examined the anti-nociceptive effects of DEX in CCI rats. Local injection to sciatic nerve with DEX inhibited the SGCs activation, NGF over-expression and sympathetic sprouting during the development of neuropathic pain. Conflict of interest The authors declare no competing interests. Acknowledgements The present work was supported by grants from Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (PPZY2015A066) and Xuzhou Science and Technology Plan Projects (KC14SH075).
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Fig.1.SGCs activation and expression levels of NGF were increased in CCI rats. (A)Immunofluorescence analysis indicated an increase in GFAP in the DRG from day1 to day14 after CCI surgery. Scale Bar=100 μm. (B) Double immunofluorescence labelling of GFAP with NGF. The result indicated that NGF could be expressed by SGCs. Scale bars=50 μm. (C) Western blot analysis showed expression levels of NFG were increased in DRG after CCI. *P<0.05 versus Sham group, * * p<0.01 versus Sham group.
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Fig.2. (A) Sprouting sympathetic after CCI. An increase of basket formations in DRG after CCI. Sympathetic fibers formed basket formations around some neurons (white arrow).(B)Immunofluorescence analysis indicated the increase in Iba-1 immunoreactivity in the ipsilateral lumbar spinal dorsal horn of CCI rats. Scale Bar=100 μm. *P<0.05 versus Sham group, * * p<0.01 versus Sham group.
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Fig.3. Pre-treat by injection of DEX started immediately after CCI surgery. (A)The increase in the PWL of CCI rats. (B) Pre-treat by DEX inhibited over-expression of NGF in CCI rats.(C) Pre-treat by DEX inhibited SGC activation in CCI rats. Scale Bar=100 μm. *p<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group, ＋ p<0.05 versus CCI7d+NS group.
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Fig.4. (A) DEX inhibited Sprouting sympathetic. (B) DEX inhibited microglia activation in the ipsilateral of spinal dorsal horn. Scale Bar=100 μm. *p<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group, ＋ p<0.05 versus CCI7d+NS group.
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Fig.5. Post-treat by injection of DEX after CCI surgery. (A)Heat hypersensitivity was alleviated by sciatic nerve block of DEX. (B) There was no significant difference in NGF expression between CCI14d+NS group and CCI14d+DEX group. (C) Post-treat by DEX showed no significant effect on SGC activation. Scale Bar=100 μm. *P<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group.
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Fig.6. (A) Post-treatment could not inhibit NGF over-expression. (B) Post-treatment could not reverse microglia activation in the ipsilateral of spinal dorsal horn. Scale Bar=100 μm. *P<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group.
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S0361-9230(17)30121-1 http://dx.doi.org/doi:10.1016/j.brainresbull.2017.04.016 BRB 9210
To appear in:
Brain Research Bulletin
Received date: Accepted date:
1-3-2017 27-4-2017
Please cite this article as: Jing-ru Wu, Hui Chen, Deng-xin Zhang, Kai Jiang, Ying-ying Yao, Ming-ming Zhang, Bo Zhou, Jie Wang, Local injection to sciatic nerve of DEX reduces pain behaviors, SGCs activation, NGF expression and sympathetic sprouting in CCI rats, Brain Research Bulletinhttp://dx.doi.org/10.1016/j.brainresbull.2017.04.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Local injection to sciatic nerve of DEX reduces pain behaviors, SGCs activation, NGF expression and sympathetic sprouting in CCI rats Jing-ru Wua,1, Hui Chena,1, Deng-xin Zhangb, Kai Jiangc, Ying-ying Yaoa,, Ming-ming Zhang a,, Bo Zhoua, Jie Wanga,*
a Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu, China, 221002 b Department of Anesthesiology, The Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China, 214000 c Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China, 215000
*Corresponding author: Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu, China, 221002 Fax:+86 15895208059 E-mail addresses: [email protected] (J. Wang) 1
These two authors contributed equally to this work.
Other authors E-mail address: [email protected] (J.-r.Wu), [email protected] (H. Chen), [email protected](D.-x. Zhang), [email protected] (K. Jiang), [email protected] (Y.-y. Yao), [email protected](M.-m. Zhang), [email protected] (B.Zhou)
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Highlights We examined the anti-nociceptive effects of DEX on peripheral nervous system. Local injection to sciatic nerve with DEX can inhibited the heat hypersensitivity. Pre-treat by DEX inhibited the pathological changes in DRGs. Post-treat by DEX had no significant effect on the induced pathological changes.
Abstract Neuropathic pain has become an intractable health threat, with its profound effect on quality of life. Dorsal root ganglia (DRG) is evidenced to play a crucial role in neuropathic pain. The peripheral nociceptive afferents seem to be essential not only to initiate the process of neuropathic pain, but also to maintain and modulate it. Dexmedetomidine (DEX), a highly selective agonist of α2-adrenergic receptor (α2-AR), has provided significant analgesia in neuropathic pain. In the present study, we found that local injection to sciatic nerve of DEX alleviated heat hypersensitivity induced by chronic constriction injury (CCI). Western blotting revealed that DEX inhibited the overexpression of nerve growth factor (NGF) significantly. Immunohistofluorescence results showed that DEX inhibited glia cells activation and sympathetic sprouting simultaneously in DRG. Our study suggests that DEX attenuates neuropathic pain in CCI rats by down-regulation of satellite glial cell (SGC) activation, NGF expression and sympathetic sprouting.
Abbreviations: DRG, dorsal root ganglia; DEX, dexmedetomidine; α2-AR, α2-adrenergic receptor; CCI, chronic constriction injury; NGF, nerve growth factor; SGC, satellite glial cell; PWL , paw withdrawal latency; GFAP, glial fibrillary acidic protein. Keywords: Neuropathic pain; Dexmedetomidine; Satellite glial cell; NGF; sympathetic sprouting; microglia
1. Introduction Numerous studies have shown that peripheral nerve injury is often accompanied by neuropathic pain. The development and maintenance of neuropathic pain are dependent on nociceptive input. Dorsal root ganglia (DRG) is an important part of peripheral nervous system. Pathological changes in DRG are critical to neuropathic pain. DRG contains primary sensory neurons, which is the first stage of nociceptive information input. After peripheral nerve injury, primary sensory neurons receive peripheral afferent information and pass it to the spinal cord dorsal horn, completing the delivery of nociceptive information (Manzano et al.,2008). -2-
Satellite glial cell (SGC) is the main type glia cell in DRG. In respond to peripheral nerve injury, the resting SGCs converted to an activated state through a series of cellular and molecular modifications (Hanani, 2005). By releasing inflammatory cytokines, chemokines and other factors known to facilitate pain signalling, SGCs play important roles in mediating neuropathic pain. It was reported that there is an increase of nerve growth factor (NGF) in DRG following peripheral nerve injury. Further study showed NGF mRNA and protein levels within the SGCs are elevated (Sebert and Shooter,1993; Wells et al.,1994; Zhou et al.,1999). NGF is widely distributed in the central and peripheral nervous system. NGF is the member of the neurotrophin family, which is essential for the survival and growth of sympathetic and sensory neurons(Lindsay,1994; Patel,2000). Studies found that NGF was involved in the pathophysiology of pain in many neuropathic pain models (Choi and Son, 2011; Belrose et al., 2014; Richards and Mcmahon, 2013). Evidence implicate that NGF is a peripheral pain mediator by working as a sensitizer of nociceptors (Crowley et al.,1994; Frade and Barde,1998; Campenot,1987; Woolf et al.,1994). Some studies showed that NGF over-expression evoked nociceptive behavior in animals and humans (Lewin and Mendell,1996; Lewin and Mendell,1994; Pertens et al.,1999; Malin et al.,2006). Experimental blockade of NGF can decrease afferent nerve activity, nociceptor sensitivity, therefore preventing hyperalgesia (Djouhri et al.,2001; McMahon et al.,1995; Lewin and Mendell,1996). By affecting the release of inflammatory mediators, promoting the growth of nerve fibers and binding with tyrosine kinase(TrkA) receptor for regulating ion channels and molecular signals, NGF plays an important role in the pathophysiology of pain (Richardson and Mcmahonsb, 2013). Dexmedetomidine (DEX) is a highly selective α2-AR agonist which has been extensively employed in anaesthesia and critical care medicine for its anxiolytic, sedative and analgesic effects (Kamibayashi and Maze, 2000; Coursin, 2001; Paris and Tonner, 2005). Numerous studies have showed that DEX exhibits a significant analgesic effect in models of chronic pain (Guneli et al., 2007; Kimura et al., 2012; Liu et al., 2012). DEX stimulates α2-AR in the locus coeruleus and spinal cord providing sedation and analgesia (Sonohata et al., 2004). Our study was designed to investigate whether DEX has an effect on peripheral nervous system in model of CCI rats.
2. Experimental procedures 2.1 Animals Male Sprague-Dawley rats (200–250g) from the experimental animal center(Xuzhou Medical University, China), housed at a constant ambient temperature of 24 ± 1 ◦C under a 12 h light/dark cycle with ad libitum access to food and water.All experiments were conducted in accordance with the guidelines of the International Association for the Study of Pain and were approved by the Committee for the Ethical Use of Laboratory Animals, Xuzhou Medical University. 2.2 Experimental groups -3-
Part1: The rats were randomly divided into 2 groups (n=28): Sham group and CCI group. Animal behavior was tested on -1, 1, 3, 7, 14 days . Part2: This experiment addressed the role of DEX on the neuropathic pain. Rats received Local injection to sciatic nerve of DEX (25μg/kg) or equal dose of NS daily as from the end of the operation to day 6. Rats were randomly divided into 4 groups(n=12): Sham+NS, Sham+DEX, CCI7d+NS and CCI7d+DEX. Animal behavior was tested on -1, 3, 5,7 days after surgery. Part3:In this part, rats were randomly divided into 4 groups(n=12): Sham+NS, Sham+DEX, CCI14d+NS and CCI14d+DEX and received local injection to sciatic nerve of DEX (25μg/kg) or equal dose of NS daily as from day 7 to day 13. Animal behavior was tested on -1, 5, 7, 10, 12,14 days . 2.3 CCI and local injection to sciatic nerve model The CCI model was created as described by Bennett and Xie(Bennett and Xie,1988). Animals were anesthetized with 10 % chloral hydrate (300 mg/kg, i.p.), and the left sciatic nerve was exposed. Four loose ligatures were made around the nerve using 4–0 braided silk thread (Ethicon Inc., Brussels,Belgium) at intervals of 1 mm. The incision was closed in layers. In sham-operated rats, the same procedure was performed without sciatic nerve ligation. According to the method reported by Leszczynska and Kau(Leszczynska and Kau,1992),rats were anesthetized with Sevoflurane, drugs were injected into the area of the popliteal fossa of the left hind limb by hamilton syringe. The rats will be checked for motor impairments after drug injection. 2.4 Behavioral test Behavioral test was performed 1 hour later after drug injection. The behavioral testing was performed by observers who were blinded to the experimental conditions. To assess paw withdrawal latency(PWL), rats were placed on glass floor of a thermal apparatus (IITC Plantar Analgesia Meter,IITC Life Science Inc)30 min for accommodation before the PWL assessment. A light of variable intensity could be applied to the plantar hindpaws.Each rat was tested five times alternately, allowing sufficient time between tests to allow paw temperatures to return to baseline.25s was set as the cutoff time to avoid tissue damage. 2.5 Immunofluorescence Method After behavioural testing, the rats were subjected to deep anesthesia with 10 % chloral hydrate (400 mg/kg, i.p.) and transcardial perfusion with 0.01 MPBS (200 mL, pH 7.4) followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB; 300 mL,pH 7.4) for fixation. The L4-5 segments of the spinal cord and DRG were removed and post-fixed for 4–6 h at 4◦C, then immersed in 30% sucrose in phosphate buffer at 4 ◦C. The tissues were embedded in optimal cutting temperature compound at -20◦C and sectioned on a cryostat (Leica CM1950,Germany). Transverse spinal sections were cut at the thickness of 35μm and The ganglia were sectioned with a vibratome at a thickness of 30μm, along the long axis of the DRG, The sections were washed with 0.3 % Triton -4-
X-100 for 15min and incubated with donkey serum for 2 h at room temperature. Spinal slices were incubated with goat anti-Iba-1(1:300; Abcam, USA).Half of the ganglia slices were incubated with rabbit anti-NGF(1:200;Abcam,USA)and mouse antiGFAP(1:400;Cell Signaling Technology,USA),other slices were incubated with rabbit anti-NeuN(1:400;Cell Signaling Technology,USA)and mouse anti-Tyrosine Hydroxylase(1:300; Millipore, Bedford, MA, USA).All the slices were then incubated at 4°C for 24 hours. After three rinses in PBS-T, the spina sections were incubated for 2 hours at room temperature in Alexa Fluor 594 donkey anti-goat IgG(1:200; Invitrogen, Carlsbad, CA, USA),and the ganglia sections were incubated in Alexa 488 donkey antimouse IgG(1:200; Invitrogen, Carlsbad, CA, USA) and Alexa 594 donkey anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA, USA). Finally, after these slices rinsed, adhered onto the microslide. Each microslide was examined under the confocal laser microscope (FV1000; Olympus, Tokyo, Japan). 2.6 Western Blotting After the behavioral tests. L4-5 DRG of the rats were isolated and stored at -80 ◦C. The ganglias and 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 ◦C, the supernatant was collected for Western blotting. Equal amounts of protein (30 μg) were loaded in each lane and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Themembranes were then sealed with the addition of 5 % skim milk in Tris-buffered saline (TBS–Tween) for 1 h at room temperature, followed by incubation with rabbit anti-NGF(1:800; Abcam, USA) and rabbit anti-β-actin (1:1000; ZSGB-Bio, China) primary anti-body at 4 ◦C overnight. The membranes were rinsed in Washing Buffer for 3 × 10 min. This was followed by incubation for 2 h at room temperature with a peroxidase-conjugated goat anti-rabbit IgG antibody (1:1000; Beyotime, China). The membranes were washed 3 × 10 min in TBS-T. Immunoreactivities were visualized by NBT/BCIP assay kit (Sigma-Aldrich). Finally, Western blot densitometry analysis of signal intensity was performed using ImageJ software. 2.7 Statistical analysis The data were represented as mean ± SEM. Statistical analysis was performed using Prism 6.0(GraphPad) software. One-way ANOVA for data from different groups and two-way ANOVA with on repeated factor for data of PWL were employed. p < 0.05 was considered statistically significant. All of the tests including behavior, Western blot and immunoflurorescence were performed blinded.
3. Results 3.1 CCI induced changes of SGCs activation, NGF expression and sympathetic sprouting in DRG In DRG sections of CCI rats, glial fibrillary acidic protein (GFAP) -5-
immunofluorescence intensity in SGCs encircling the neurons was measured as indication of activation. The results showed that GFAP level increased as early as day 1 after CCI. The increase was significant on day 7 and 14, as compared to the Sham group (p<0.05; Fig.1A). The NGF level also increased after CCI, and is co-localized with GFAP activation in SGC (Fig.1B). Western blotting confirmed the increase of NGF was significant from day 3 to day 14 of CCI (p<0.05; Fig.1C). To quantify sympathetic sprouting after the nerve lesion, basket formations around some neurons was measured in fixed sections by immunostaining for tyrosine hydroxylase. The results showed increase of basket formations beginning on day 3 after CCI versus the Sham group (p<0.05; Fig.2A). 3.2 CCI induced microglia activation in the spinal dorsal horn In the spinal cord, the intensity of ionized calcium binding adapter molecule 1 (Iba-1, a microglia marker) was measured and normalized. The results indicated that the fluorescence intensity of Iba-1 in the ipsilateral spinal dorsal horn increased significantly after CCI surgery versus the Sham group (p<0.05; Fig.2B). 3.3 Effect of pre-treatment by local injection to sciatic nerve with DEX after CCI surgery Rats received local injection to sciatic nerve (DEX 25 μg/kg) or equal dose of NS daily after surgery until day 6. Behavioral test was performed 1 hour later after the injection.The results showed there were no significant differences in PWL between Sham+NS and Sham+DEX groups (p>0.05). Compared with the Sham groups, PWL in CCI7d+NS group was significantly decreased (p<0.05). The PWL of CCI7d+DEX group was significantly increased versus CCI7d+NS group, but still lower than the Sham groups (p<0.05)(Fig.3A). Intensity of GFAP and Iba-1, protein expression of NGF and basket formations in CCI7d+DEX group was significantly lower than those in the CCI+NS group (p<0.05), but still greater than those in the Sham groups (p<0.05). The results showed the injections from day 0 to day 6 significantly inhibited the glia activation,up-expression of NGF and sprouting of sympathetic fibers (Fig.3B-C;Fig.4A-B). 3.4 Effect of post-treatment by local injection to sciatic nerve with DEX after CCI surgery In this part, rats received local injection to sciatic nerve of DEX (25 μg/kg) or an equal dose of NS daily as from day7 to day13. Compared with Sham groups, the PWL in CCI14d+DEX group was significantly decreased from day10 to day14, but showed a significant increase than CCI14d+NS group (p<0.05; Fig.5A). Compared with CCI14d+NS group,the intensity of GFAP and Iba-1, protein expression of NGF and basket formations in CCI14d+DEX group had no significant differences (p>0.05; Fig.5B-C;Fig.6A-B).
4. Discussion In the group without DEX, the PWL in CCI rats decreased from as early as day1 and maintained at a low level throughout the experiment. We found CCI surgery alone induced SGCs activation, followed by NGF up-regulation and sympathetic sprouting at -6-
DRG. CCI surgery also induced microglia activation in the spinal dorsal horn. These results are consistent with previous studies. Pre-treat by local injection to sciatic nerve of DEX after CCI surgery for 7 consecutive days was effective in preventing heat hypersensitivity and related pathological changes. Post-treat by local injection to sciatic nerve of DEX can alleviate heat hypersensitivity, but had no significant effect on the induced pathological changes. SGCs are glial cells wrapped around the primary sensory neurons in the peripheral nervous system. Though much less abundant than astrocytes and microglia in the spinal cord, SGCs are the main type of glia cell in the DRG(Hanani, 2010). Due to its unique location in the sensory ganglia, SGCs are capable of exerting a strong influence on pain transmission. In respond to nerve injury, SGCs upregulate GFAP protein expression, undergo cell division and release various algesic substances (Elson et al., 2004b; Zhang et al., 2009). SGCs express a series of receptors which can respond to the neurotransmitters released by neurons. By participating in the exchange of information among neurons, these neurotransmitters play important roles in the control of neuronal microenvironment and mediation of neuronal excitability (Weick et al., 2003; Ceruti et al.,2008).Activated SGCs generate and release inflammatory cytokines and neurotransmitters such as bradykinin, interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α), neurotrophins, and ATP into its surroundings. SGC modulate pain by influencing neighboring neuron’s function and status with those released chemicals (Suadicani et al., 2010; Takeda et al., 2008; Zhang et al., 2007). NGF,which can be released from SGC is one of the most important molecular mediators of peripheral sensitization (Crowley et al., 1994; Frade and Barde, 1998; Campenot, 1987). NGF regulates expression of various pain-related proteins in nociceptive sensory neurons, including substance P, Navl.8 sodium channel, brainderived neurotrophic factor (BDNF), etc. These proteins further sensitize nociceptive neurons, promoting the activation of secondary neurons in the central nervous system (Mantyhpw et al., 2011；Balaratnasingam and Janca，2012；Schnegelsberg et al., 2010; Nishigami et al., 2013). NGF participates in mediating inflammatory and immune responses after peripheral nerve injury (Herzberg et al., 1997). By activating mast cells, neutrophils and eosinophils, releasing a variety of pain mediators such as 5-HT, prostaglandins, bradykinin, IL-1β, TNF-α, histamine and other mediators of inflammation, amplifying the inflammatory response, NGF stimulates the nociceptors and promotes the generation of pain (Schnegelsbergb et al.,2010;Nishigamit et al., 2013). NGF mediates ion channel opening such as those expressed on sensory neurons the sodium channel Nav1.8, which is involved in NGF-induced thermal hyperalgesia (Kerr, 2001). Evidence shows that NGF modulates this sodium channel current through the protein kinase A pathway following TrkA binding (Brackenbury, 2007). Since many NGF-responsive neurons contain the vanilloid receptor TRPV1, it is suspected that this receptor plays a role in NGF-mediated hypersensitivity (Ng et al., 2011). Stimulations cause TRPVl opening and Ca2+ influx, generating action potentials in nociceptive neurons for pain signal conduction (Ramsey et al., 2006). Studies have shown that NGF increases binding of NGF to TrkA receptors on nociceptive neurons activated -7-
phospholipase C and reduced TRPVl opening threshold, thus inducing neurons sensitivity. NGF increased TRPVl expression and promoted its insertion into the plasma membrane (Donnerer et al., 2005; Xue et al., 2007; Zhang et al., 2005). We speculate that by these NGF reduces the action potential (AP) threshold of nociceptive neurons. Under normal physiological conditions, the sympathetic nervous system is not directly associated with peripheral nerve sensory. NGF up-regulation is concomitant with an increase in overall sympathetic fiber density in the cellular region of the DRG, contributing to the appearance of “basket’’ formations (Chien et al., 2005; Pertin et al., 2007). NGF alters expression of ion channels and receptors, as the result of sensory neurons remodelling contributes to the hypersensitive to noradrenaline released by Sympathetic fibers(McLachlan et al., 1993; Xie et al., 2010; Zhang and Tan,2011). The abnormal sympathetic-sensory coupling and the sympathetic sprouting involved in the formation and modulation of neuropathic pain will cause paresthesia or hyperalgesia (Pertin et al., 2007; Xie et al., 2007). DRG is an important part of the coupling after peripheral nerve injury. Microglia activation is dependent on nociceptive input. After nerve injury, the sensory neurons activate microglia by releasing neurotransmitters and inflammatory mediators. Microglia activation mainly manifests as morphological changes: limb-like protrusion disappears and becomes amoeba-like, accompanied by specific cell marker expression and enhanced cell proliferation. By autocrine and paracrine, its activation is further strengthened(Grace et al., 2011). Our results showed that the microglial marker Iba-1 was significantly upregulated until to day 14 after CCI surgery. Activated microglia produces and releases large amounts of inflammatory cytokines, modulating neuronal excitability. Activated microglia can act on the surrounding non-activated astrocytic to induce inflammation and immune responses. These lead to hyperalgesia and allodynia(Griffin et al., 2007; Tsuda et al., 2005; Scholz and Woolf, 2007). In current study, DEX inhibits of the microglia activation. Like previous reports, nerve injury increases SGCs and microglia activation and that many pain treatments reduce such increases. This effect could result from the reduction of nociception input by the drug, not by the drug itself. Multiple studies have demonstrated that systemic or intrathecal administration of DEX produces significant anti-nociceptive effects in models of neuropathic pain.DEX produces its effect by stimulating α2-ARs in sensory neurons. Activation of this receptor leads to a series of effects, such as reduction of cyclic AMP, protein kinase A and Na+ channels activation. Attenuation of hyperalgesia by DEX is dependent on the activation of α2-ARs on the presynaptic membrane of neurons. DEX reduced the release of pronociceptive neurotransmitters, such as glutamate from the primary afferent terminals, hyperpolarized spinal interneurons, resulting in suppression of pain signals transmission (Poree et al., 1998). Study showed DEX affected the locus coeruleus and was dependent on the noradrenergic pathways. By inducing presynaptic membrane depolarization and inhibiting the release of substance P, NE, or other “harmful” peptides, DEX suppressed the transmission of nociceptive stimulation in the spinal dorsal horn (Nelson et al., 2003). In peripheral nervous system, DEX inhibited APs conduction by depressing voltage-gated Na+-channel currents (Oda et al., 2007). Furthermore, it has -8-
been reported that systemic administration of DEX, which suppresses the expression of mRNAs encoding inflammatory cytokines in inflammatory cells, inhibiting the amplification of inflammatory response (Kanazi et al., 2006). Some studies showed mix DEX with local anesthetics can enhance its effectiveness, shortening sensory and motor block onset and extended the analgesia duration(Memis ¸et al., 2004; Kanazi et al., 2006). Due to this, we chose DEX given perineurally in the current study. Pre-treat by local injection to sciatic nerve of DEX after nerve injury alleviated heat hypersensitivity significantly. In DRG,DEX inhibited SGCs activation, NGF over-expression and sympathetic sprouting. The above results suggest inhibition of spontaneous activity occurred with the use of DEX, as evidenced by the reduction of peripheral input through inhibition of microglia activation in spinal dorsal horn. However, local injection to sciatic nerve from day 7 after surgery showed little effect as compared with those which started DEX immediately after the nerve injury. This interesting result suggests that SGCs activation and subsequent pathological changes are irreversible, and treatment should follow the principle of preemptive analgesia, i.e. The treatment should begin at the initial stages of the pain. Once neuropathic pain has already been established, it is much more difficult to reverse the biological and biochemical changes in the activated cells. In the current study, the rats receiving DEX didn’t show motor impairments, as evidenced by little difference in heat hypersensitivity among Sham+NS and Sham+DEX groups. This shows that DEX does not produce a nerve blockade as local anesthetics. DEX produces its effect probably by stimulating α2-ARs in sensory neurons, reducing its reaction to damage, enhancing the ability of anti-nociception. DEX increases the AP threshold of nociceptive neurons, reducing the reaction to neurotransmitters and inflammatory cytokines. Stimulating α2-ARs can inhibit the upregulation of some receptors and sodium channels in sensory neurons. DEX may also inhibit the generation and release of inflammatory cytokines and chemokines by SGCs. In summary, in the present study, we examined the anti-nociceptive effects of DEX in CCI rats. Local injection to sciatic nerve with DEX inhibited the SGCs activation, NGF over-expression and sympathetic sprouting during the development of neuropathic pain. Conflict of interest The authors declare no competing interests. Acknowledgements The present work was supported by grants from Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (PPZY2015A066) and Xuzhou Science and Technology Plan Projects (KC14SH075).
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Fig.1.SGCs activation and expression levels of NGF were increased in CCI rats. (A)Immunofluorescence analysis indicated an increase in GFAP in the DRG from day1 to day14 after CCI surgery. Scale Bar=100 μm. (B) Double immunofluorescence labelling of GFAP with NGF. The result indicated that NGF could be expressed by SGCs. Scale bars=50 μm. (C) Western blot analysis showed expression levels of NFG were increased in DRG after CCI. *P<0.05 versus Sham group, * * p<0.01 versus Sham group.
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Fig.2. (A) Sprouting sympathetic after CCI. An increase of basket formations in DRG after CCI. Sympathetic fibers formed basket formations around some neurons (white arrow).(B)Immunofluorescence analysis indicated the increase in Iba-1 immunoreactivity in the ipsilateral lumbar spinal dorsal horn of CCI rats. Scale Bar=100 μm. *P<0.05 versus Sham group, * * p<0.01 versus Sham group.
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Fig.3. Pre-treat by injection of DEX started immediately after CCI surgery. (A)The increase in the PWL of CCI rats. (B) Pre-treat by DEX inhibited over-expression of NGF in CCI rats.(C) Pre-treat by DEX inhibited SGC activation in CCI rats. Scale Bar=100 μm. *p<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group, ＋ p<0.05 versus CCI7d+NS group.
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Fig.4. (A) DEX inhibited Sprouting sympathetic. (B) DEX inhibited microglia activation in the ipsilateral of spinal dorsal horn. Scale Bar=100 μm. *p<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group, ＋ p<0.05 versus CCI7d+NS group.
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Fig.5. Post-treat by injection of DEX after CCI surgery. (A)Heat hypersensitivity was alleviated by sciatic nerve block of DEX. (B) There was no significant difference in NGF expression between CCI14d+NS group and CCI14d+DEX group. (C) Post-treat by DEX showed no significant effect on SGC activation. Scale Bar=100 μm. *P<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group.
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Fig.6. (A) Post-treatment could not inhibit NGF over-expression. (B) Post-treatment could not reverse microglia activation in the ipsilateral of spinal dorsal horn. Scale Bar=100 μm. *P<0.05 versus Sham+NS group, # p<0.05 versus Sham+DEX group.
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