CXCR3 contributes to neuropathic pain via ERK activation in the anterior cingulate cortex
Jing Qin, Ang Li, Yan Huang, Run-Hua Teng, Yan Yang, Yong-Xing Yao
a Department of Anesthesia, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, PR China
b Department of Anesthesia, Tinglin Hospital of Jinshan District, Shanghai, PR China
c Department of Anesthesia, the Central Hospital of Lishui City, Lishui, PR China
d Zhejiang Chinese Medical University, Hangzhou, PR China
e Centre for Neuroscience, Zhejiang University School of Medicine, Hangzhou, PR China
A B S T R A C T
The anterior cingulate cortex (ACC) is activated by noxious stimuli and is involved in the affective component of pain processing; but its role in the sensory component of pain remains largely unknown. Studies have verified that Chemokine (C-X-C motif) receptor 3 (CXCR3) is involved in nociceptive sensitization in the spinal cord after peripheral nerve injury; however, the expression of CXCR3 in the ACC and its role in neuropathic pain has not been reported. Here, we showed that CXCR3 co-localized with neurons in the ACC and the upregulation of CXCR3 corresponded with hypersensitive behaviors after a chronic constriction injury of the sciatic nerve. Pharmacological blockade of CXCR3 using local injection of its inhibitor, AMG487, into the ACC significantly attenuated hyperalgesia induced by chronic constriction injury and suppressed the phosphorylation of extracellular signal-regulated kinase (ERK). Collectively, these results suggest that CXCR3 in the ACC is involved in hyperalgesia induced by pe- ripheral nerve injury and ERK may be a downstream target.
1. Introduction
Neuropathic pain (NP) is a direct consequence of a lesion or disease of the somatosensory system [1]. Due to the high preva- lence, debilitating effects, and high social cost, NP has become a public health problem [2,3]. Since little is known about the path- ogenesis of NP, most treatments rely on pharmacological in- terventions, such as anti-depressants, calcium channels blockers, and opioids [4,5]; however, these drugs are not curative and often produce several side effects. Given that previous NP studies have focused mostly on the spinal cord, research into the supraspinal mechanisms is still lacking.
The ACC is part of the limbic system and forms the frontal part of the cingulate cortex [6], which is located on the inner side of the cerebral hemispheres [7]. Unlike other cortical regions that consist of layers I-VI, in which each layer contains specific interneurons,pyramidal cells and projection fibers, the ACC lacks layer IV [6,8]. The synapses in ACC are highly plastic, making it a heterogeneous cortex that receives afferent input from other parts of the nervous system and integrates information within different layers [9,10]. In some models of NP, chemical or electrolytic lesions of the ACC alleviate the emotional-affective component of pain [11,12], while not affecting the sensory-discriminative component of pain [13].
Chemokine (C-X-C motif) receptor 3 (CXCR3) is associated with various neurological diseases, including multiple sclerosis and Alzheimer’s disease [14]. In recent years, a number of studies have proven that CXCR3 is elevated in the spinal cord and DRGs of several rodent NP models [15]. Additionally, pharmacological blockade or genetic knockdown of CXCR3 can alleviate NP [16,17], indicating that CXCR3 has a vital role in chronic pain. However, the expression and distribution patterns of CXCR3 in the ACC as well as its involvement in NP, has not been explored.
Extracellular signal-regulated kinase (ERK) plays an important role in synaptic plasticity [18] and central sensitization of pain [19,20]. In the ACC, ERK is involved in synaptic potentiation [21] and its activation contributes to the development and maintenance of NP [22,23]. Furthermore, ERK has been identified as a potential downstream target of CXCR3 in the spinal cord [15,24]. Intrathecaladministration of the CXCR3 inhibitor, AMG487, not only attenuates hyperalgesia, but also suppresses ERK phosphorylation induced by NP model in rats [15]. However, it is unclear if the CXCR3/ERK signaling pathway as well as the antihyperalgesic effects of AMG487 exist in the ACC.
In the present study, we investigated: 1) the expression and distribution of CXCR3 in the ACC; 2) its role in NP; 3) if AMG487 has antihyperalgesic effects in the ACC; and 4) the interaction between CXCR3 and ERK in the ACC and its role in pain modulation.
2. Material and methods
2.1. Animals
Male Sprague-Dawley rats (weight 220 ± 20 g) were obtained from the Experimental Animal Center of Zhejiang Academy of Medical Sciences. Rats were housed in groups (3e4 rats/cage) at atemperature of 24 ± 2 ◦C in a 12 h light/dark cycle, with ad libitumaccess to food and water. The experimental protocol was approved by the Research Ethics Committee of the First Affiliated Hospital at Zhejiang University and complied with the internationally accredited guidelines and ethical regulations on animal research. Efforts were made to minimize the number and suffering of animals.
2.2. Induction of neuropathic pain
Rats were randomly divided into 2 groups, a sham and CCI group. In the sham group, the left sciatic nerve was exposed, but not ligated; in the CCI group, the left sciatic nerve was ligated, as pre- viously described by Bennett and Xie [25]. Briefly, the rats were anesthetized with isoflurane, after which the left sciatic nerve was ligated using three strands of 4-0 chromic gut sutures placed 1 mm apart from each other. After surgery, the rat was given a subcu- taneous injection of penicillin (0.5 mL/rat, 96 mg/mL) to prevent infections.
2.3. Drug administration
AMG487 was purchased from Med Chem Express (USA) and dissolved in 10% dimethyl sulfoxide (DMSO). Rats were randomly assigned into 3 groups: CCI þ DMSO (1 mL), CCI þ AMG487 (2.5 mg/1 mL), or CCI AMG487 (5 mg/1 mL). After anesthetizing the rat with4% pentobarbital sodium (0.15 mL/100 g), the head was fixed in the prone position on the stereotactic apparatus to expose bregma. Two holes (depth, 0.2 mm) were drilled on each side (bregma forward, 1.7 mm; lateral, 0.6 mm) of the cranium and a trocar (Shenzhen Ruiwode Life Science and Technology Co., LTD, Guang- dong, China) was inserted. The rats were then given a subcutaneousinjection of penicillin to prevent infections. CCI was performed on day 7 after catheterization. AMG487 were bilaterally (0.5 mL each side) injected into the ACC on days 5, 6, and 7 after CCI surgery.
2.4. Behavioral tests
2.4.1. Mechanical paw withdrawal threshold
The mechanical paw withdrawal threshold (MWT) was deter- mined using von Frey filaments. Rats were acclimatized for 30 min in a plastic box (12 cm 15 cm 22 cm) elevated on a wire mesh grid. As described by Fox [26], the plantar surface was stimulated with filaments of increasing stiffness (0.4 ge26 g) and we assessed positive responses, such as a quick withdrawal or licking of the paw. The testing was repeated 3 times with at least 5 min between stimulations and the average value was taken as the final MWT.
2.4.2. Acetone test score
Cold sensitivity was tested using the acetone test score (ATS), as described by Choi et al. [27] and was conducted using the same apparatus as the MWT test. Briefly, 50 mL of acetone was sprayed onto the plantar surface and we observed the responses for 20 s after application. The results were scored on a 3-point scale: 0 points: no response; 1 point: slight paw withdrawal; 2 points: fierce paw withdrawal; 3 points: shaking the paw or screeching. The test was repeated 3 times with at least 5 min between appli- cations and the average value was taken as the final ATS.
2.5. Western blot
After deeply anesthetizing the rats with pentobarbital sodium, we decapitated the rat, removed the brain, and divided ACC tissue into the left (CCI) and right (contralateral) sides for protein analysis. Protein samples were separated using SDSepolyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. The membrane was blocked in 5% skim milk at room temperature for 1 h, after which it was incubated in the following primary anti-bodies at 4 ◦C for 48 h: CXCR3 (1:500, Abcam, Cambridge, UK), ERK(1:2000, Cell Signaling Technology, Danvers, USA), PERK (1:2000, Cell Signaling Technology), and GAPDH (1:10,000, Kang Chen Bio- tech, Shanghai, China). After washing the primary antibody with TBST, we incubated the membranes in HRP-conjugated secondary antibody for 2 h at room temperature. The immune complexes were detected by an enhanced chemiluminescence reagent (Thermo Fisher, Waltham, MA, USA) and captured using a Chem- iDoc MP System (Bio-Rad, Hercules, CA, USA).
2.6. Immunofluorescence assay
Rats were anesthetized with pentobarbital sodium and trans- cardially perfused with saline, followed by 4% paraformaldehyde. After perfusion, the ACC was harvested, post-fixed with 4% paraformaldehyde for 48 h, and then dehydrated with 30% sucrose for 3 days at 4 ◦C. Transverse ACC sections (30 mm) were blocked with 10% sheep serum for 2 h at room temperature and then incubated with the following primary antibodies for 48 h at 4 ◦C: rabbit-anti-Iba1 (1:1000, Abcam), rabbit-anti-GFAP (1:500, Abcam), rabbit-anti-NeuN (1:2000, Abcam), and mouse-anti-CXCR3 (1:800, Santa Cruz Biotechnology, Dallas, TX, USA). The sections were then rinsed with PBS thrice and incubated with FITC-conjugated goat anti- rabbit-IgG (1:200, Proteintech, Rosemont, IL, USA) and CY3- conjugated goat anti-mouse-IgG (1:200, Proteintech) in the dark for 2 h at room temperature. After, the sections were then exam- ined with a fluorescence microscope (x-cite 120; OLYMPUS, Japan).
2.7. Statistical analysis
All data are expressed as mean ± SEM and were analyzed using Prism 5.0 (GraphPad Software). Behavioral differences among the 3 groups were analyzed using a two-way analysis of variance (ANOVA) and unpaired Student’s t-tests for comparisons between two groups. Western blot data were analyzed using a one-way ANOVA. The significance level was set at p < 0.05.
3. Results
3.1. Expression and distribution of CXCR3 in the ACC
Immunofluorescence experiments were conducted to explore the expression and cellular distribution of CXCR3 in the ACC. We performed double immunofluorescence staining for CXCR3 with NeuN (a neuronal marker), GFAP (an astrocytic marker), and Iba-1 (a microglial marker). Merged signal indicated that CXCR3 was predominantly co-localized with NeuN, but not with astrocytes or microglia in the ACC (Fig. 1).
3.2. CCI-induced hyperalgesia and CXCR3 overexpression in the ACC
To validate our neuropathic pain model, the MWT and ATS were assessed before and after CCI surgery. The baseline MWT and ATS were not significantly different between the sham and CCI groups. On day 7 post-surgery, the MWT of the ipsilateral paw in the CCI group was significantly lower than that in the sham group (n ¼ 4,p < 0.001; Fig. 2A). Conversely, the ATS of the ipsilateral paw in the CCI group was significantly higher than that in the sham group (n 4, p < 0.001, Fig. 2B). To investigate a potential role of CXCR3 in the ACC in NP, we detected the abundance of CXCR3 in the ACC using Western blot. Compared to the sham group, expression of CXCR3 was bilaterally higher in the CCI group (n 4, CCI vs sham, p < 0.05; contra vs sham, p < 0.01; Fig. 2C).
3.3. AMG487 attenuated hyperalgesia and suppressed ERK phosphorylation
To further investigate the role of CXCR3 in the initiation of NP, the selective CXCR3 inhibitor, AMG487, was bilaterally injected into the ACC on days 5, 6, and 7 following CCI surgery. The MWT and ATS were measured 2 h after the last drug administration. Compared to DMSO, AMG487 significantly increased the MWT in a dose- dependent manner (n 6, p < 0.001; Fig. 3A). Similarly, AMG487 significantly decreased the ATS (n 6, p < 0.001; Fig. 3B). No sig- nificant differences were observed in the baseline MWT and ATS values between the CCI DMSO and CCI AMG487 groups. These findings indicate that AMG487 can attenuate CCI-induced hyper- sensitivity. To further explore the molecular mechanisms of the antihyperalgesic effects of AMG487, we used Western blot to measure the levels of total ERK and phosphorylated ERK abundance in the ACC. Bilateral injections of AMG487 dose-dependently sup- pressed the phosphorylation of ERK in the ACC compared to that of the CCI DMSO group (n 4, p < 0.001; Fig. 3D); however, AMG487 did not affect the total ERK abundance in the ACC (Fig. 3C).
4. Discussion
In the present study, we demonstrated that CXCR3 is expressed in the neurons, but not astrocytes or microglia, of the ACC. Addi- tionally, we showed that the abundance of CXCR3 bilaterally increased 7 days following CCI surgery, consistent with the corre- sponding hypersensitive behaviors. Microinjection of the CXCR3 inhibitor, AMG487, significantly attenuated CCI-induced hyper- algesia, indicating that CXCR3 in the ACC contributes to the NP associated with peripheral nerve injury. Furthermore, AMG487 administration also suppressed the phosphorylation of ERK in a dose-dependent manner, which suggests that ERK could be a downstream target of CXCR3 in the ACC.
The sensory-discriminative component of pain is mainly inte- grated by the primary somatosensory cortex, while the emotional- affective component is synthetically integrated by the secondary somatosensory cortex, insula, and ACC [28]. The ACC is part of the limbic system, is activated by noxious stimuli, and is involved in pain processing, especially affective pain [9]. Interestingly, in our study, a pharmacological approach successfully attenuated CCI- induced hyperalgesia in a dose-dependent manner, which is con- tradictory to the traditional view that the ACC is associated with the regulation of the emotional-affective, but not the sensory- discriminative component, of pain. Consistent with this, studies have found that optogenetic activation of specific neurons in the ACC reduces CFA- and formalin-induced hypersensitive behaviors [29,30]. It has been reported that anatomically, the direct projection of the ACC to the spinal dorsal horn [31] and periaqueductal grey[32] is involved in the descending modulation of spinal paintransmission and specifically, is activated in NP [33]. Therefore, this positive feedback loop (spinal dorsal horn e ACC e spinal dorsalhorn) may have been disrupted by the pharmacological approach in our study; however, the exact mechanisms warrant further investigation.
Chemokines play an important role in chemotaxis, regulation of inflammation, and pain in the nervous system by activating the seven transmembrane domain G-protein coupled receptors (GPCRs) [34]. Several recent studies have reported that CXCR3, a chemokine receptor, is expressed in the central nervous system and is involved in some neurological diseases [35,36]. Furthermore, CXCR3 in the spinal cord and DRGs have a substantial role in the hypersensitive behaviors induced by nerve injury; consistent with this, pharmacological blockade or knockdown of CXCR3 signifi- cantly alleviates this hyperalgesia [17,37]. However, CXCR3 expression in the ACC and its role in NP have not been previously explored. We found that CXCR3 exclusively co-localized with neurons, but not astrocytes or microglia, in the ACC; this was different from spinal cord expression, in which CXCR3 is expressed in neurons, astrocytes, and microglia [24]. This discrepancy sug- gests that the distribution of CXCR3 is tissue-specific, involving different physiological or pathological mechanisms. To address how CXCR3 in the ACC is involved in pain modulation, we utilized the CCI neuropathic pain model and consistent with previous studies, CCI successfully induced mechanical hypersensitivity [19,38] and cold allodynia. While previous studies have found that CXCR3 is upregulated in the spinal cord only on the ipsilateral side to injury [16], we found that CXCR3 was upregulated on both the ipsilateral and contralateral sides of the ACC. Although the cause of this discrepancy is currently unknown, this could be due to anatomical differences between the spinal cord and ACC. To further explore how CXCR3 in the ACC effects pain modulation and knowing that chemokines cause local effects, we performed ACC microinjections of the selective CXCR3 inhibitor, AMG487, on days 5, 6, and 7 following CCI surgery. Tests for MWT and ATS on day 7 revealed that AMG487 significantly attenuated CCI-induced hyperalgesia, indicating that CXCR3 in the ACC plays a vital role in pain modulation after peripheral nerve injury.
Pyramidal and non-pyramidal cells are the two primary neuronal cell types in the ACC [9]. Pyramidal neurons express AMPA and NMDA receptors on their membranes and are activated by various somatic and visceral noxious stimuli [39]; non- pyramidal cells primarily express GABA receptors and usually actas inhibitory interneurons [29]. Activation of AMPA and NMDA receptors in the ACC have been shown to increase Ca2þ concen- tration in the neuronal cytoplasm, which then activates the ERK in phosphorylated way [40,41]. Interestingly, recent studies haveshown that activation of CXCR3 in DRGs can also increase Ca2þ concentration in the cytoplasm [42] and lead to ERK activation [43].
In the ACC, ERK activation contributes to both the development and maintenance of chronic pain [22]. In our study, the phosphorylation of ERK in the ACC was suppressed by AMG487 in a dose-dependent manner; this is consistent with several studies in which intrathecal administration of AMG487 attenuates NP in an ERK-dependent manner [15,24]. There are two potential mechanisms of the anti- hyperalgesic effects of AMG487 in the ACC: 1) AMG487 acts directly on CXCR3, which then leads to the inhibition of ERK phosphory- lation or 2) AMG487 indirectly interrupts NMDA or AMPA receptor function via direct effects on CXCR3, which then inhibits ERK phosphorylation. Electrophysiological activation of CXCR3 in the spinal cord increases spontaneous excitatory postsynaptic as wellas NMDA and AMPA receptor-induced activity [16]. Previous studies have shown that CXCR5 knockdown in the ACC alleviates negative emotions associated with NP by regulating NMDA and AMPA receptors on pyramidal neurons [44]. It is possible that the antihyperalgesic effects of AMG487 was achieved through a similar molecular mechanism.
In conclusion, CXCR3 contributes to NP induced by peripheral nerve injury through modulation of ERK phosphorylation in the ACC. These findings suggest that CXCR3 in the ACC could be a therapeutic target for NP.
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