CXCR4 is a recently reported chemokine receptor that plays a pivotal role in mediating neuropathic pain . In the present study, we first demonstrated that CXCR4 expression significantly increased in compressed DRG from CCD rats. Knockdown of CXCR4 remarkably attenuated CCD-induced multiple pain behaviors, including mechanical and thermal hyperalgesia, mechanical and cold allodynia, suggesting that CXCR4 contributed CCD-induced low back pain. Moreover, we showed that intervertebral foramen injection of plerixafor, a CXCR4 selective antagonist, was able to reverse CCD-induced pain behaviors by reducing CCD-induced Nav1.8 and Nav1.9 up-regulation in compressed DRG. We further found that the Nav1.8 selective blocker, A-803467 and a non-selective VGSCs blocker, ropivacaine cold both relieve CCD-induced pain behaviors after intervertebral foramen injection, but the analgesic duration was apparently shorter than that of plerixafor. Finally, we demonstrated that intervertebral foramen injection of plerixafor eased CCD-induced pain due to its local effect on compressed DRG rather than the systemic effects, and the approach of intervertebral foramen plerixafor injection has no significant effect on the basal pain sensitivity. Taken together, these findings suggested that chemokine receptor CXCR4 involved in CCD-induced pain by up-regulating Nav1.8 and Nav1.9 expression, while intervertebral foramen plerixafor injection is a novel and appropriate therapeutic strategy to treat low back pain.
4.1 CXCR4-Nav1.8/Nav1.9 axis contributes to CCD-induced multiple pain behaviors
Low back pain is a major health problem over the world which is becoming worse largely because of the increase in the population base and the intensification of aging . Despite the huge medical investment in the low back pain, its global burden is still heavy. During the past few decades, the clinical management of low back pain remains unsatisfactory. Commonly, low back pain closely related to the intraforaminal stenosis, laterally herniated disk, vertebral fracture, or inﬂammatory disorders . To develop an effective treatment, it is important to understand the underlying mechanism of low back pain, which necessarily promoted a number of preclinical models of low back pain being developed. In the present study, we used the CCD model induced by inserting a stainless steel rod between vertebrae holes (L4 and/or L5) which has been proven to be a typical model that mimics the clinical etiology and various symptoms of low back pain3. In line with previous studies, we identified four different pain behaviors in the ipsilateral hindpaw of CCD rats, including mechanical hyperalgesia, thermal hyperalgesia, mechanical allodynia and cold allodynia [22, 29], which occurred as early as 1 day after surgery and maintained at least for 14 days.
Using CCD model, we found that the expression level of CXCR4 in compressed DRG dramatically increased revealed by immunofluorescent staining which was in accordance with a recent study . The functional nature of the up-regulated CXCR4 was identified by our siRNA knockdown assay in which intraganglionar microinjection with CXCR4 siRNA obviously attenuated CCD-induced multiple pain behaviors. Additionally, blockade of CXCR4 with intervertebral foramen plerixafor injection showed the ability to reverse CCD-induced pain behaviors. All these results demonstrated that activation of CXCR4 in compressed DRG contributed to CCD-induced pain. As supporting, we previously have verified that intraganglionar microinjection with CXCR4 siRNA was able to relieve complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain and spared nerve injury (SNI)-induced chronic neuropathic pain . Moreover, the CXCR4 are found to be related with inflammatory pain-induced DRG neuronal hyperexcitability by our previous study in which plerixafor could inhibit the increased firing rate and reverse the lowered action potential rheobase . Interestingly, it was reported that the DRG neuronal excitability extremely enhanced and the voltage-gated sodium currents in small and medium-sized DRG neurons also increased after compression [19, 21, 30]. It was well known that VGSCs were very important for electrogenesis and nerve impulse conduction, especially Nav1.8 which provided a significant contribution to action potential generation and Nav1.9 which contribute to setting the resting membrane potential and to neuronal excitability . Here, we found for the first time that the expression of Nav1.8 and Nav1.9 in the compressed DRG neurons dramatically increased and the A-803467, a selective Nav1.8 blocker, as well as the local anesthetic ropivacine was efficient to alleviate CCD-induced pain behaviors after intervertebral foramen injection, which strongly supported that Nav1.8 and Nav1.9 were involved in the pathogenesis of low back pain. Our further study showed that intervertebral foramen plerixafor injection was sufficient to reducing the enhanced expression of Nav1.8 and Nav1.9 accompanying with easing pain behaviors, suggesting that the Nav1.8 and Nav1.9 were the downstream effector of CXCR4 activation for pain genesis. Actually, the contribution of CXCR4 activation-induced DRG neuronal protein synthesis to pain has been indicated in our previous study in which the protein translation inhibitor could block the acute mechanical hyperalgesia evoked by intraplantar injection of CXCL12, the ligand of CXCR4 . In addition, we further have demonstrated that CXCL12-CXCR4 signaling could regulate the excitability of primary nociceptive neurons by up-regulating Nav1.8 expression and contribute to persistent pain and hyperalgesia induced by intraplantar bee venom injection . Taking together, these findings evidenced that CXCR4-Nav1.8/Nav1.9 axis in compressed DRG was involved in CCD-induced low back pain. Recently, another study demonstrated that NaV1.6 and regulatory NaVβ4 sodium channel in compressed DRG also play key roles in low back pain . Besides, TRPV4, voltage-gated potassium current and hyperpolarization-activated cation current were all reported to be correlated with CCD-induced low back pain [30, 32, 33]. Whether these changed ion channels and currents shared the same upstream trigger mechanism (CXCR4 activation) remains unknown which are definitely needed to be explored in the future experiments.
4.2 Blocking CXCR4 with plerixafor is a potential and efficient treatment for neuropathic pain
Neuropathic pain is considered to be the most intractable chronic pain with current therapeutic approaches being unsatisfactory and inefficient. Thus, it is urgent to explore the new potential targets for pain management. CXCR4 is a seven transmembrane G protein-coupled receptor (GPCR) which has been widely concerned in the field of neuropathic pain research . It is constitutively distributed in the somatosensory and nociceptive system . In consistent with a recent study, we previously found that CXCR4 present in both non-peptidergic (IB4-positive) and peptidergic (SP-positive or CGRP-positive) primary nociceptor neurons and also co-localized with TRPV1, a thermonociceptor of primary sensory afferent . In the central nervous system (CNS), spinal dorsal horn and thalamus as well as somatosensory cortex which are the primary centers for pain perception have a high level basal expression of CXCR4 . All these findings strongly suggested that CXCR4 is closely related to pain. Here, we identified that the expression level of CXCR4 in normal DRG was at a low level, while it extremely enhanced under CCD state. In support of our finding, emerging evidence have demonstrated that CXCR4 expression in the nervous system was inducible under neuropathic pain state. In diabetic condition, the total number of DRG neurons expressing CXCR4 mRNA significantly increased comparing to normal DRG . In chronic constriction injury (CCI) and SNI model, elevation of CXCR4 immunoreactivity has also been demonstrated in the DRG neuronal soma [36, 37]. The expression level of CXCR4 in the CNS could also be obviously enhanced under central neuropathic pain situations such as central post stroke pain , spinal cord ischemia-reperfusion , spinal cord contusion injury . This inducible expression pattern of CXCR4 enable CXCR4 as an ideal analgesic target. Actually, extensive behavioral pharmacology experiments have proven that blocking CXCR4 with its specific antagonist, plerixafor, was sufficient to prevent and reverse various neuropathic pain . Importantly, plerixafor is hematopoietic stem cell mobilizer often used in clinic with its safety and efficacy has been approved by the U.S. Food and Drug Administration in 2008 . It is noteworthy that intervertebral foramen plerixafor injection did not affect the basal pain response in normal rat, indicating that plerixafor could selectively relieve pathological pain without affecting the normal sensory or motor function. Additionally, we found that the analgesic duration of intervertebral foramen single plerixafor injection lasted at least for 24 h which was much longer than ropivacaine, a kind of local anesthetics commonly used for nerve blocking in clinic. We speculated that the protracted analgesic effect was largely because of the plerixafor not only reduced the expression of Nav1.8 and Nav1.9, but also has the ability to relieve inflammation in compressed DRG which was also a pivotal mechanism for neuronal hyperexcitability and chronic pain. In supporting, recent studies have demonstrated that the pro-inflammatory cytokines dramatically up-regulated accompanying with several anti-inflammatory cytokines down-regulated in compressed DRG , while plerixafor has been proven to be able to reducing the expression of TNF-α, IL-1β, IL-6 and inflammatory cell infiltration in the nervous system [10, 35]. Collectively, the above characteristics and advantages of plerixafor make it an effective and applicable therapeutic medicine for neuropathic pain.
4.3 Intervertebral foramen injection is an appliable treatment approach for low back pain
As we known, many drugs used to treat neuropathic pain are delivered systematically and intrathecally which has a major strategic limitation. In addition to on-target effects, systemic delivery of these drugs has side effects because of off-target neural suppression in the peripheral nervous system (PNS), and even more so in the CNS. For plerixafor, the common adverse effects in clinical trials were dizziness, nausea and diarrhea which were observed in over 10% of patients, largely because CXCR4 is widely distributed in nervous system and presented in various kinds of cells . An alternative approach for treating pain is delivering drugs into the DRG which has been recognized that DRG is a critical structure in sensory transduction and modulation, including pain transmission and the maintenance of persistent pain states. There are multiple changes in gene expression in DRG under pain condition, encompassing a large number of distinct family members including neuropeptides, receptors, ion channels, signal transduction molecules, synaptic vesicle proteins, and others [41, 42]. The functional consequence of these changes is sensitization and hyperexcitability of DRG neurons, which then leads to persistent pain . Moreover, the DRG is consistently locating at the intervertebral neural foramina within a capsule which is an easily accessible target. All these unique characteristics make the DRG an ideal target for the treatment of chronic pain . Clinically, the local anesthetics and steroid were usually injecting into the intervertebral foramina to block the electrogenesis and pain conduction at DRG level to cure various kinds of pain with the assistance of visual equipment, like the ultrasound and computed tomography . In the present study, we provided preclinical evidence that intervertebral foramina injection at the compressed DRG of plerixafor or local anesthetics was sufficient to reverse the CCD-induced multiple pain behaviors. However, intervertebral foramina injection at the adjacent non-compressed DRG did not affect the pain behaviors, suggesting that the plerixafor dose for intervertebral foramina injection has no systemic effects. All of these data strongly indicated that intervertebral foramen plerixafor injection is an appliable treatment approach for low back pain.