Spinal CCK contributes to somatic hyperalgesia induced by orofacial inflammation combined with stress in adult female rats.

In some chronic primary pain conditions such as temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS), mild or chronic stress enhances pain. TMD and FMS often occur together, but the underlying mechanisms are unclear. The purpose of this study was to investigate the role of cholecystokinin (CCK) in the spinal cord in somatic hyperalgesia induced by orofacial inflammation combined with stress. Somatic hyperalgesia was detected by the thermal withdrawal latency and mechanical withdrawal threshold. The expression of CCK1 receptors, CCK2 receptors, ERK1/2 and p-ERK1/2 in the spinal cord was examined by Western blot. After the stimulation of orofacial inflammation combined with 3 day forced swim, the expression of CCK2 receptors and p-ERK1/2 protein in the L4-L5 spinal dorsal horn increased significantly, while the expression of CCK1 receptors and ERK1/2 protein remained unchanged. Intrathecal injection of the CCK2 receptor antagonist YM-022 or mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 blocked somatic hyperalgesia induced by orofacial inflammation combined with stress. Intrathecal administration of the MEK inhibitor blocked somatic sensitization caused by the CCK receptor agonist CCK8. The CCK2 receptor antagonist YM-022 significantly reduced the expression of p-ERK1/2. These data indicate that upregulation of CCK2 receptors through the MAPK pathway contributes to somatic hyperalgesia in this comorbid pain model. Thus, CCK2 receptors and MAPK pathway may be potential targets for the treatment of TMD comorbid with FMS.

Role of innate immunity in chemotherapy-induced peripheral neuropathy.

It has become increasingly clear that the innate immune system plays an essential role in the generation of many types of neuropathic pain including that which accompanies cancer treatment. In this article we review current findings of the role of the innate immune system in contributing to cancer treatment pain at the distal endings of peripheral nerve, in the nerve trunk, in the dorsal root ganglion and in the spinal dorsal horn.

Effects of external low intensity focused ultrasound on inflammatory markers in neuropathic pain.

BACKGROUND: Changes in inflammatory cytokine levels contribute to the induction and maintenance of neuropathic pain. We have shown that external low intensity focused ultrasound (liFUS) reduces allodynia in a common peroneal nerve injury (CPNI). Here, we investigate an underlying mechanism of action for this treatment and measure the effect of liFUS on inflammatory markers. METHODS: Male rats were divided into four groups: CPNI/liFUS, CPNI/shamliFUS, shamCPNI/liFUS, and shamCPNI/shamliFUS. Mechanical nociceptive thresholds were measured using Von Frey filaments (VFF) to confirm the absence/presence of allodynia at baseline, after CPNI, and after liFUS. Commercial microarray and ELISA assays were used to assess cytokine expression in the treated L5 dorsal root ganglion (DRG) and dorsal horn (DH) tissue 24 and 72 h after liFUS. RESULTS: VFF thresholds were significantly reduced following CPNI in both groups that received the injury (p < 0.001). After liFUS, only the CPNI/liFUS cohort showed a significant increase in mechanical thresholds (p < 0.001). CPNI significantly increased TNFa, IL6, CNTF, IL1b (p < 0.05 for all) levels in the DRG and DH, compared to baseline, consistent with previous work in sciatic nerve injury. LiFUS in CPNI rats resulted in a decrease in these cytokines in DRG 72 h post-therapy (TNFa, IL6, CNTF and IL1b, p < 0.001). In the DH, IL1b, CNTF, and TNFa (p < 0.05 for all) decreased 72 h after liFUS. CONCLUSION: We have demonstrated that liFUS modifies inflammatory cytokines in both DRG and DH in CPNI rats. These data provide evidence that liFUS, reverses the allodynic phenotype, in part, by altering inflammatory cytokine pathways.

Dorsal horn disinhibition and movement-induced behaviour in a rat model of inflammatory arthritis.

OBJECTIVES: Alterations beyond joint inflammation such as changes in dorsal horn (DH) excitability contribute to pain in inflammatory arthritis (IA). More complete understanding of specific underlying mechanisms will be important to define novel targets for the treatment of IA pain. Pre-clinical models are useful, but relevant pain assays are vital for successful clinical translation. For this purpose, a method is presented to assess movement-induced pain-related behaviour changes that was subsequently used to investigate DH disinhibition in IA. METHODS: IA was induced by intra-articular injection of complete Freund's adjuvant (CFA) in male rats, and weight distribution was assessed before and after walking on a treadmill. To confirm increased activity in nociception-related pathways, fos expression was assessed in the superficial DH, including in nociceptive neurons, identified by neurokinin 1 (NK1) immunoreactivity, and interneurons. Inhibitory terminal density onto NK1+ neurons was assessed and lastly, a cohort of animals was treated for 3 days with gabapentin. RESULTS: At 4 weeks post-CFA, walking reduced weight distribution to the affected joint and increased DH fos expression, including in NK1+ neurons. Neuronal activity in inhibitory cells and inhibitory terminal density on NK1+ neurons were decreased in CFA-treated animals compared with controls. Treatment with gabapentin led to recovered behaviour and DH neuronal activity pattern in CFA-treated animals. CONCLUSION: We describe an assay to assess movement-induced pain-related behaviour changes in a rodent IA model. Furthermore, our results suggest that disinhibition may contribute to pain related to movement in IA.

Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses.

BACKGROUND: Complex regional pain syndrome type-I (CRPS-I) is a progressive and devastating pain condition. The mechanisms of CRPS-I still remain poorly understood. We aim to explore expression profiles of genes relevant to pain and neuroinflammation mechanisms involved in CRPS-I. METHODS: The rat chronic post-ischemic pain (CPIP) model that mimics human CRPS-I was established. RNA-sequencing (RNA-Seq), qPCR, Western blot, immunostaining, and pharmacological studies were used for profiling gene changes in ipsilateral spinal cord dorsal horn (SCDH) of CPIP model rat and further validation. RESULTS: CPIP rats developed persistent mechanical allodynia in bilateral hind paws, accompanied with obvious glial activation in SCDH. RNA-Seq identified a total of 435 differentially expressed genes (DEGs) in ipsilateral SCDH of CPIP rats. qPCR confirmed the expression of several representative genes. Functional analysis of DEGs identified that the most significantly enriched biological processes of upregulated genes include inflammatory and innate immune response. We further identified NLRP3 inflammasome expression to be significantly upregulated in SCDH of CPIP rats. Pharmacological blocking NLRP3 inflammasome reduced IL-1ß overproduction, glial activation in SCDH as well as mechanical allodynia of CPIP rats. CONCLUSION: Our study revealed that immune and inflammatory responses are predominant biological events in SCDH of CPIP rats. We further identified NLRP3 inflammasome in SCDH as a key contributor to the pain and inflammation responses in CPIP rats. Thus, our study provided putative novel targets that may help to develop effective therapeutics against CRPS-I.

The development of pain circuits and unique effects of neonatal injury.

Pain is a necessary sensation that prevents further tissue damage, but can be debilitating and detrimental in daily life under chronic conditions. Neuronal activity strongly regulates the maturation of the somatosensory system, and aberrant sensory input caused by injury or inflammation during critical periods of early postnatal development can have prolonged, detrimental effects on pain processing. This review will outline the maturation of neuronal circuits responsible for the transmission of nociceptive signals and the generation of pain sensation-involving peripheral sensory neurons, the spinal cord dorsal horn, and brain-in addition to the influences of the neuroimmune system on somatosensation. This summary will also highlight the unique effects of neonatal tissue injury on the maturation of these systems and subsequent consequences for adult somatosensation. Ultimately, this review emphasizes the need to account for age as an independent variable in basic and clinical pain research, and importantly, to consider the distinct qualities of the pediatric population when designing novel strategies for pain management.

The innate immune response as a mediator of osteoarthritis pain.

In this narrative review, we discuss the emerging role of innate immunity in osteoarthritis (OA) joint pain. First, we give a brief description of the pain pathway in the context of OA. Then we consider how neuro-immune signaling pathways may promote OA pain. First, activation of neuronal Pattern Recognition Receptors by mediators released in a damaged joint can result in direct excitation of nociceptors, as well as in production of chemokines and cytokines. Secondly, indirect neuro-immune signaling may occur when innate immune cells produce algogenic factors, including chemokines and cytokines, that act on the pain pathway. Neuro-immune crosstalk occurs at different levels of the pathway, starting in the joint but also in the innervating dorsal root ganglia and in the dorsal horn. Synovitis is characterized by recruitment of immune cells, including macrophages, mast cells, and CD4+ lymphocytes, which may contribute to nociceptor sensitization and OA pain through production of algogenic factors that amplify the activation of sensory neurons. We discuss examples where this scenario has been suggested by findings in human OA and in animal models. Overall, increasing evidence suggests that innate immune pathways play an initiating as well as facilitating role in pain, but information on how these pathways operate in OA remains limited. Since these innate pathways are eminently targetable, future studies in this area may provide fruitful leads towards a better management of symptomatic OA.

Transfer of complex regional pain syndrome to mice via human autoantibodies is mediated by interleukin-1-induced mechanisms.

Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin-muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic deletion of interleukin-1 (IL-1) using IL-1αß knockout (KO) mice and perioperative IL-1 receptor type 1 (IL-1R1) blockade with the drug anakinra, but not treatment with the glucocorticoid prednisolone, prevented these changes. Anakinra treatment also reversed the established sensitization phenotype when initiated 8 days after incision. Furthermore, with the generation of an IL-1ß floxed(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1ß, suggesting that both peripheral and central inflammatory mechanisms contribute to the transferred disease phenotype. These results indicate that persistent CRPS is often contributed to by autoantibodies and highlight a potential therapeutic use for clinically licensed antagonists, such as anakinra, to prevent or treat CRPS via blocking IL-1 actions.

High Omega-3 Polyunsaturated Fatty Acids in fat-1 Mice Reduce Inflammatory Pain.

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs), such as α-linolenic and linoleic acids, are essential fatty acids in mammals, because they cannot be synthesized de novo. However, fat-1 transgenic mice can synthesize omega-3 PUFAs from omega-6 PUFAs without dietary supplementation of omega-3, leading to abundant omega-3 PUFA accumulation in various tissues. In this study, we used fat-1 transgenic mice to investigate the role of omega-3 PUFAs in response to inflammatory pain. A high omega-3 PUFA tissue content attenuated formalin-induced pain sensitivity, microglial activation, inducible nitric oxide synthase expression, and the phosphorylation of NR2B, a subunit of the N-methyl-d-aspartate (NMDA) receptor. Our findings suggest that elevated omega-3 PUFA levels inhibit NMDA receptor activity in the spinal dorsal horn and modulate inflammatory pain transmission by regulating signal transmission at the spinal dorsal horn, leading to the attenuation of chemically induced inflammatory pain.

IL-17 contributed to the neuropathic pain following peripheral nerve injury by promoting astrocyte proliferation and secretion of proinflammatory cytokines.

Central neuroinflammation is important in the pathophysiological processes of neuropathic pain following peripheral nerve injury. Recently, interleukin-17 (IL-17) has been detected in different inflammatory conditions of the central nervous system and contributes to neuropathic pain associated with multiple sclerosis, experimental autoimmune encephalomyelitis. The present study, based on the rat model of spinal nerve ligation, analyzed the infiltration of cluster of differentiation (CD)4+ T cells and the expression of IL­17 in the spinal cord during the maintenance phase of neuropathic pain, and investigated central inflammatory reaction and astrocyte activation. The results demonstrated that the infiltrated CD4+ T cells in the spinal cord increased in the rat model of spinal nerve ligation, and immunofluorescence staining demonstrated that the CD4+/IL­17+ cells were located at superficial laminae of spinal dorsal horn. This was accompanied by significant upregulation of IL­17. Furthermore, the mRNA expression levels of IL­1ß and IL­6 were also significantly enhanced in model rats compared with the sham and control groups in the spinal dorsal horn. In vitro, the proliferation ability and secretion of proinflammatory cytokines notably increased in the IL­17­stimulated astrocytes. Results from the present study indicate that IL­17 may contribute to neuropathic pain by promoting the proliferation of astrocytes and secretion of proinflammatory cytokines in spinal nerve ligation­induced neuropathic pain.

Toll-like receptor 4 contributes to chronic itch, alloknesis, and spinal astrocyte activation in male mice.

Increasing evidence suggests that Toll-like receptor 4 (TLR4) contributes importantly to spinal cord glial activation and chronic pain sensitization; however, its unique role in acute and chronic itch is unclear. In this study, we investigated the involvement of TLR4 in acute and chronic itch models in male mice using both transgenic and pharmacological approaches. Tlr4 mice exhibited normal acute itch induced by compound 48/80 and chloroquine, but these mice showed substantial reductions in scratching in chronic itch models of dry skin, induced by acetone and diethylether followed by water (AEW), contact dermatitis, and allergic contact dermatitis on the neck. Intrathecal (spinal) inhibition of TLR4 with lipopolysaccharide Rhodobacter sphaeroides did not affect acute itch but suppressed AEW-induced chronic itch. Compound 48/80 and AEW also produced robust alloknesis, a touch-elicited itch in wild-type mice, which was suppressed by intrathecal lipopolysaccharide R sphaeroides and Tlr4 deletion. Acetone and diethylether followed by water induced persistent upregulation of Tlr4 mRNA and increased TLR4 expression in GFAP-expressing astrocytes in spinal cord dorsal horn. Acetone and diethylether followed by water also induced TLR4-dependent astrogliosis (GFAP upregulation) in spinal cord. Intrathecal injection of astroglial inhibitor L-α-aminoadipate reduced AEW-induced chronic itch and alloknesis without affecting acute itch. Spinal TLR4 was also necessary for AEW-induced chronic itch in the cheek model. Interestingly, scratching plays an essential role in spinal astrogliosis because AEW-induced astrogliosis was abrogated by putting Elizabethan collars on the neck to prevent scratching the itchy skin. Our findings suggest that spinal TLR4 signaling is important for spinal astrocyte activation and astrogliosis that may underlie alloknesis and chronic itch.

Neuropathic pain is constitutively suppressed in early life by anti-inflammatory neuroimmune regulation.

Peripheral nerve injury can trigger neuropathic pain in adults but not in infants; indeed, for unknown reasons, neuropathic pain is rare before adolescence. We show here that the absence of neuropathic pain response in infant male rats and mice following nerve injury is due to an active, constitutive immune suppression of dorsal horn pain activity. In contrast to adult nerve injury, which triggers a proinflammatory immune response in the spinal dorsal horn, infant nerve injury triggers an anti-inflammatory immune response, characterized by significant increases in IL-4 and IL-10. This immediate anti-inflammatory response can also be evoked by direct C-fiber nerve stimulation in infant, but not adult, mice. Blockade of the anti-inflammatory activity with intrathecal anti-IL10 unmasks neuropathic pain behavior in infant nerve injured mice, showing that pain hypersensitivity in young mice is actively suppressed by a dominant anti-inflammatory neuroimmune response. As infant nerve injured mice reach adolescence (postnatal day 25-30), the dorsal horn immune profile switches from an anti-inflammatory to a proinflammatory response characterized by significant increases in TNF and BDNF, and this is accompanied by a late onset neuropathic pain behavior and increased dorsal horn cell sensitivity to cutaneous mechanical and cold stimuli. These findings show that neuropathic pain following early life nerve injury is not absent but suppressed by neuroimmune activity and that "latent" pain can still emerge at adolescence, when the neuroimmune profile changes. The data may explain why neuropathic pain is rare in young children and also why it can emerge, for no observable reason, in adolescent patients.

Spinal neuroimmune activation is independent of T-cell infiltration and attenuated by A3 adenosine receptor agonists in a model of oxaliplatin-induced peripheral neuropathy.

Many commonly used chemotherapeutics including oxaliplatin are associated with the development of a painful chemotherapy-induced peripheral neuropathy (CIPN). This dose-limiting complication can appear long after the completion of therapy causing a significant reduction in quality-of-life and impeding cancer treatment. We recently reported that activation of the Gi/Gq-coupled A3 adenosine receptor (A3AR) with selective A3AR agonists (i.e., IB-MECA) blocked the development of chemotherapy induced-neuropathic pain in models evoked by distinct agents including oxaliplatin without interfering with their anticancer activities. The mechanism(s) of action underlying these beneficial effects has yet to be explored. Our results herein demonstrate that the development of oxaliplatin-induced mechano-hypersensitivity (allodynia and hyperalgesia) in rats is associated with the hyperactivation of astrocytes, but not microglial cells, increased production of pro-inflammatory and neuroexcitatory cytokines (TNF, IL-1ß), and reductions in the levels of anti-inflammatory/neuroprotective cytokines (IL-10, IL-4) in the dorsal horn of the spinal cord. These events did not require lymphocytic mobilization since oxaliplatin did not induce CD45(+)/CD3(+) T-cell infiltration into the spinal cord. A3AR agonists blocked the development of neuropathic pain with beneficial effects strongly associated with the modulation of spinal neuroinflammatory processes: attenuation of astrocytic hyperactivation, inhibition of TNF and IL-1ß production, and an increase in IL-10 and IL-4. These results suggest that inhibition of an astrocyte-associated neuroinflammatory response contributes to the protective actions of A3AR signaling and continues to support the pharmacological basis for selective A3AR agonists as adjuncts to chemotherapeutic agents for the management of chronic pain.

Oligodendrocyte ablation triggers central pain independently of innate or adaptive immune responses in mice.

Mechanisms underlying central neuropathic pain are poorly understood. Although glial dysfunction has been functionally linked with neuropathic pain, very little is known about modulation of pain by oligodendrocytes. Here we report that genetic ablation of oligodendrocytes rapidly triggers a pattern of sensory changes that closely resemble central neuropathic pain, which are manifest before overt demyelination. Primary oligodendrocyte loss is not associated with autoreactive T- and B-cell infiltration in the spinal cord and neither activation of microglia nor reactive astrogliosis contribute functionally to central pain evoked by ablation of oligodendrocytes. Instead, light and electron microscopic analyses reveal axonal pathology in the spinal dorsal horn and spinothalamic tract concurrent with the induction and maintenance of nociceptive hypersensitivity. These data reveal a role for oligodendrocytes in modulating pain and suggest that perturbation of oligodendrocyte functions that maintain axonal integrity can lead to central neuropathic pain independent of immune contributions.