Maintenance Of Pain Hypersensitivity Research Articles

Spinal microglia-derived TNF promotes the astrocytic JNK/CXCL1 pathway activation in a mouse model of burn pain

Burn injury-induced pain (BIP) is an extremely complicated condition usually resistant to analgesic drugs, while its pathogenesis remains unknown. Considerable attention has been attracted to elucidate the glial mechanisms in chronic pain. In this study, we initiatively used a mouse model of second‐degree BIP to investigate the underlying non-neuronal mechanisms at the spinal cord level. Our behavioral results showed that hind-paw burn injury caused persistent allodynia and hyperalgesia for 2 weeks in mice. Further studies revealed that both microglia and astrocytes activated in a spatially- and temporally-dependent manner in spinal cord after burn injury. In addition, the phosphorylated p38 mitogen-activated protein kinase (MAPK)-mediated tumor necrosis factor (TNF) release in spinal microglia is essentially attributed to the early stage of BIP, while the c-Jun N-terminal kinase (JNK) MAPK-dependent chemokine CXCL1 expression is mainly involved in the maintenance of pain hypersensitivity. Most strikingly, burn injury-induced pain symptoms and the activation of astrocytes were significantly suppressed by TNF inhibitor Thalidomide. On the contrary, intrathecal injection of TNF caused apparent pain hypersensitivity, accompanied by the activation of astrocytes and the upregulation of CXCL1 via the JNK MAPK signaling pathway, indicating that TNF is the key cytokine in the interaction between microglia and astrocytes at the spinal level. Moreover, treatment with the CXCR2 receptor antagonist SB225002 to block the biological activities of CXCL1 significantly attenuated the mechanical allodynia and thermal hyperalgesia in this BIP model. Taken together, this study indicates that intervention of glial pathways provides a new perspective in the management of BIP.

Sex differences in neuroimmune and glial mechanisms of pain.

Pain is the primary motivation for seeking medical care. Although pain may subside as inflammation resolves or an injury heals, it is increasingly evident that persistency of the pain state can occur with significant regularity. Chronic pain requires aggressive management to minimize its physiological consequences and diminish its impact on quality of life. Although opioids commonly are prescribed for intractable pain, concerns regarding reduced efficacy, as well as risks of tolerance and dependence, misuse, diversion, and overdose mortality rates limit their utility. Advances in development of nonopioid interventions hinge on our appreciation of underlying mechanisms of pain hypersensitivity. For instance, the contributory role of immunity and the associated presence of autoimmune syndromes has become of particular interest. Males and females exhibit fundamental differences in innate and adaptive immune responses, some of which are present throughout life, whereas others manifest with reproductive maturation. In general, the incidence of chronic pain conditions, particularly those with likely autoimmune covariates, is significantly higher in women. Accordingly, evidence is now accruing in support of neuroimmune interactions driving sex differences in the development and maintenance of pain hypersensitivity and chronicity. This review highlights known sexual dimorphisms of neuroimmune signaling in pain states modeled in rodents, which may yield potential high-value sex-specific targets to inform future analgesic drug discovery efforts.

Spinal microglia-neuron interactions in chronic pain.

Current deficiency in our understanding of acute-to-chronic pain transition remains a hurdle for developing effective treatments against chronic pain. Whereas neurocentric mechanisms alone are insufficient to provide satisfactory explanation for such transition, neuro-immune crosstalk has attracted attention in recent pain research. In contrast to brain microglia, spinal microglia are activated immediately in various pain states. The fast-responsive enrichment and activation of spinal microglia among different pain conditions have highlighted the crucial role of neuroinflammation caused by microglia-neuron crosstalk in pain initiation. Recent studies have revealed spinal microglia-neuron interactions are also involved in chronic pain maintenance, albeit, with different anatomic distribution, cellular and molecular mechanisms, and biologic functions. Delineating the exact temporal discrepancies of spinal microglia distribution and functions along acute-to-chronic pain transition may provide additional mechanistic insights for drug development to prevent deterioration of acute pain into the chronic state. This narrative review summerizes the longitudinal alterations of spinal microglia-neuron interactions in the initiation of pain hypersensitivity, acute-to-chronic pain progression, and chronic pain maintenance, followed by an overview of current clinical translation of preclinical studies on spinal microglia. This review highlights the crucial role of the interaction between spinal microglia and neighboring neurons in the initiation and maintenance of pain hypersensitivity, in relation to the release of cytokines, chemokines, and neuroactive substances, as well as the modulation of synaptic plasticity. Further exploration of the uncharted functions of spinal microglia-neuron crosstalk may lead to the design of novel drugs for preventing acute-to-chronic pain transition.

Characterization of the SIM-A9 cell line as a model of activated microglia in the context of neuropathic pain.

Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.

BIX01294, a G9a inhibitor, alleviates nerve injury-induced pain hypersensitivities during both development and maintenance periods.

Genetic knockdown or knockout of the histone methytransferase G9a in the injured dorsal root ganglion (DRG) has been shown to alleviate neuropathic pain development. However, the application of genetic strategy in clinic is highly limited. The present study sought to examine the effect of intrathecal BIX01294, a specific G9a inhibitor, on the development and maintenance of pain hypersensitivities caused by unilateral L5 spinal nerve injury (SNL) or chronic constriction injury (CCI) to the sciatic nerve in rats. We found that intrathecal administration of BIX01294 reduced SNL- or CCI-induced mechanical allodynia, thermal hyperalgesia and cold allodynia not only in the development period but also in the maintenance period. These effects were dose-dependent. Intrathecal administration of BIX01294 also blocked the SNL-induced increase in the level of H3K9me2, a marker of G9a activity, and reversed SNL-induced downregulation of Oprm1 mRNA, Oprk1 mRNA, Oprd1 mRNA, Kcna2 mRNA, and Kcna4 mRNA, the downstream targets of G9a, in the ipsilateral L5 DRG. These findings further implicate that G9a as a potential target in the management of neuropathic pain.

Contributions of p38 and ERK to the antinociceptive effects of TGF-β1 in chronic constriction injury-induced neuropathic rats.

BackgroundTransforming growth factor-βs (TGF-βs) are a group of multifunctional proteins that have neuroprotective roles in various experimental models. We previously reported that intrathecal (i.t.) injections of TGF-β1 significantly inhibit neuropathy-induced thermal hyperalgesia, spinal microglia and astrocyte activation, as well as upregulation of tumor necrosis factor-α. However, additional cellular mechanisms for the antinociceptive effects of TGF-β1, such as the mitogen-activated protein kinase (MAPK) pathway, have not been elucidated. During persistent pain, activation of MAPKs, especially p38 and extracellular signal-regulated kinase (ERK), have crucial roles in the induction and maintenance of pain hypersensitivity, via both nontranscriptional and transcriptional regulation. In the present study, we used a chronic constriction injury (CCI) rat model to explore the role of spinal p38 and ERK in the analgesic effects of TGF-β1.MethodsWe investigated the cellular mechanisms of the antinociceptive effects of i.t. injections of TGF-β1 in CCI induced neuropathic rats by spinal immunohistofluorescence analyses.ResultsThe results demonstrated that the antinociceptive effects of TGF-β1 (5 ng) were maintained at greater than 50 % of the maximum possible effect in rats with CCI for at least 6 h after a single i.t. administration. Thus, we further examined these alterations in spinal p38 and ERK from 0.5 to 6 h after i.t. administration of TGF-β1. TGF-β1 significantly attenuated CCI-induced upregulation of phosphorylated p38 (phospho-p38) and phosphorylated ERK (phospho-ERK) expression in the dorsal horn of the lumbar spinal cord. Double immunofluorescence staining illustrated that upregulation of spinal phospho-p38 was localized to neurons, activated microglial cells, and activated astrocytes in rats with CCI. Additionally, increased phospho-ERK occurred in activated microglial cells and activated astrocytes. Furthermore, i.t. administration of TGF-β1 markedly inhibited phospho-p38 upregulation in neurons, microglial cells, and astrocytes. However, i.t. injection of TGF-β1 also reduced phospho-ERK upregulation in microglial cells and astrocytes.ConclusionsThe present results demonstrate that suppressing p38 and ERK activity affects TGF-β1-induced analgesia during neuropathy.

Consistent sex-dependent effects of PKMζ gene ablation and pharmacological inhibition on the maintenance of referred pain.

BackgroundPersistently active PKMζ has been implicated in maintaining spinal nociceptive sensitization that underlies pain hypersensitivity. However, evidence for PKMζ in the maintenance of pain hypersensitivity comes exclusively from short-term studies in males using pharmacological agents of questionable selectivity. The present study examines the contribution of PKMζ to long-lasting allodynia associated with neuropathic, inflammatory, or referred visceral and muscle pain in males and females using pharmacological inhibition or genetic ablation.ResultsPharmacological inhibition or genetic ablation of PKMζ reduced mild formalin pain and slowly developing contralateral allodynia in nerve-injured rats, but not moderate formalin pain or ipsilateral allodynia in models of neuropathic and inflammatory pain. Pharmacological inhibition or genetic ablation of PKMζ also effectively reduced referred visceral and muscle pain in male, but not in female mice and rats.ConclusionWe show pharmacological inhibition and genetic ablation of PKMζ consistently attenuate long-lasting pain hypersensitivity. However, differential effects in models of referred versus inflammatory and neuropathic pain, and in males versus females, highlight the roles of afferent input-dependent masking and sex differences in the maintenance of pain hypersensitivity.

Optogenetic Silencing of Nav1.8-Positive Afferents Alleviates Inflammatory and Neuropathic Pain.

We report a novel transgenic mouse model in which the terminals of peripheral nociceptors can be silenced optogenetically with high spatiotemporal precision, leading to the alleviation of inflammatory and neuropathic pain. Inhibitory archaerhodopsin-3 (Arch) proton pumps were delivered to Nav1.8+ primary afferents using the Nav1.8-Cre driver line. Arch expression covered both peptidergic and nonpeptidergic nociceptors and yellow light stimulation reliably blocked electrically induced action potentials in DRG neurons. Acute transdermal illumination of the hindpaws of Nav1.8-Arch+ mice significantly reduced mechanical allodynia under inflammatory conditions, while basal mechanical sensitivity was not affected by the optical stimulation. Arch-driven hyperpolarization of nociceptive terminals was sufficient to prevent channelrhodopsin-2 (ChR2)-mediated mechanical and thermal hypersensitivity in double-transgenic Nav1.8-ChR2+-Arch+mice. Furthermore, prolonged optical silencing of peripheral afferents in anesthetized Nav1.8-Arch+ mice led to poststimulation analgesia with a significant decrease in mechanical and thermal hypersensitivity under inflammatory and neuropathic conditions. These findings highlight the role of peripheral neuronal inputs in the onset and maintenance of pain hypersensitivity, demonstrate the plasticity of pain pathways even after sensitization has occurred, and support the involvement of Nav1.8+ afferents in both inflammatory and neuropathic pain. Together, we present a selective analgesic approach in which genetically identified subsets of peripheral sensory fibers can be remotely and optically inhibited with high temporal resolution, overcoming the compensatory limitations of genetic ablations.

Downregulation of miR-219 enhances brain-derived neurotrophic factor production in mouse dorsal root ganglia to mediate morphine analgesic tolerance by upregulating CaMKIIγ.

BackgroundIncreasing evidence suggests that microRNAs are functionally involved in the initiation and maintenance of pain hypersensitivity, including chronic morphine analgesic tolerance, through the posttranscriptional regulation of pain-related genes. We have previously demonstrated that miR-219 regulates inflammatory pain in the spinal cord by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ). However, whether miR-219 regulates CaMKIIγ expression in the dorsal root ganglia to mediate morphine tolerance remains unclear.ResultsMiR-219 expression was downregulated and CaMKIIγ expression was upregulated in mouse dorsal root ganglia following chronic morphine treatment. The changes in miR-219 and CaMKIIγ expression closely correlated with the development of morphine tolerance, which was measured using the reduction of percentage of maximum potential efficiency to thermal stimuli. Morphine tolerance was markedly delayed by upregulating miR-219 expression using miR-219 mimics or downregulating CaMKIIγ expression using CaMKIIγ small interfering RNA. The protein and mRNA expression of brain-derived neurotrophic factor were also induced in dorsal root ganglia by prolonged morphine exposure in a time-dependent manner, which were transcriptionally regulated by miR-219 and CaMKIIγ. Scavenging brain-derived neurotrophic factor via tyrosine receptor kinase B-Fc partially attenuated morphine tolerance. Moreover, functional inhibition of miR-219 via miR-219-sponge in naive mice elicited thermal hyperalgesia and spinal neuronal sensitization, which were both suppressed by CaMKIIγ small interfering RNA or tyrosine receptor kinase B-Fc.ConclusionsThese results demonstrate that miR-219 contributes to the development of chronic tolerance to morphine analgesia in mouse dorsal root ganglia by targeting CaMKIIγ and enhancing CaMKIIγ-dependent brain-derived neurotrophic factor expression.

Inhibition of spinal ERK1/2–c-JUN signaling pathway counteracts the development of low doses morphine-induced hyperalgesia

Morphine-induced hyperalgesia is a pharmacological phenomenon often hindering its prolonged applications in the clinic. It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. Extracellular signal-regulated kinase (ERK) pathway contributes to pain sensitization, and its phosphorylation under pain conditions results in the induction and maintenance of pain hypersensitivity. The present study was designed to determine whether low dose morphine treatment in mice could influence the spinal activity of ERK. The data showed that morphine (1µg/kg) induced a marked increase in ERK phosphorylation. Intrathecal pre-treatment with a selective mitogen-activated and extracellular signal-regulated kinase (MEK) inhibitor PD98059, attenuated morphine-associated thermal hyperalgesia. Morphine exposure increased phosphorylation of c-JUN, that was prevented by the inhibition of ERK pathway. In addition, double immunofluorescence studies revealed that, p-ERK and p-c-JUN are localized on neurons of the spinal dorsal horn expressing µ receptors. These data suggest that ERK contributes to the morphine-induced hyperalgesia by regulating the activation of c-JUN.

Effects of surgery and/or remifentanil administration on the expression of pERK1/2, c-Fos and dynorphin in the dorsal root ganglia in mice

Tissue injury and/or opioids induce plastic changes in the spinal cord resulting in pain hypersensitivity; the contribution of the dorsal root ganglia (DRG) is poorly understood. We evaluated DRG phenotypic changes induced by surgery and/or remifentanil in a mice model of postoperative pain using as neuronal markers ERK1/2 and c-Fos; prodynorphin mRNA and dynorphin levels were also determined. We hypothesized that a correlation between nociception and DRG reactivity would occur. Surgery and/or remifentanil induced mechanical hypersensitivity, correlated with ERK1/2 phosphorylation and c-Fos expression in the DRG; changes were greater in the remifentanil + incision group and still present on day 14 (p < 0.01 vs. control). Intrathecal PD98059 (ERK1/2 inhibitor) partially reversed the mechanical hypersensitivity (44%, p < 0.05) observed in the remifentanil + incision group. In this group, significant increases in prodynorphin mRNA (at 2, 7, and 14days, p < 0.01) roughly coincided with increases in dynorphin (days 2 and 14, p < 0.001) in the DRG. Remifentanil or incision (alone) also induced an up-regulation in prodynorphin mRNA expression on days 7 and 14 (p < 0.01, p < 0.05, respectively), partially correlating with dynorphin levels. On day 21, all molecular changes returned to control levels in all experimental conditions, concurring with the complete recovery of nociceptive thresholds. Surgery and/or remifentanil induce up-regulation of c-Fos and pERK in the DRG, approximately correlating with nociceptive behavior, also associated with an increased expression of prodynorphin/dynorphin. These changes support the role of the DRG in the development and maintenance of pain hypersensitivity after surgery. The findings could contribute to the development of new therapeutic agents focused on peripheral targets.

Dorsal Horn α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Trafficking in Inflammatory Pain

Activation of synaptic N-methyl-D-aspartic acid receptor and its intracellular downstream signals in dorsal horn neurons of spinal cord contribute to central sensitization, a mechanism that underlies the development and maintenance of pain hypersensitivity in persistent pain. However, the molecular process of this event is not understood completely. Recently, new studies suggest that peripheral inflammatory insults drive changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit trafficking via N-methyl-D-aspartic acid receptor-triggered activation of protein kinases in dorsal horn and raise the possibility that such changes might contribute to central sensitization in persistent pain. This review presents current evidence regarding the changes that occur in the trafficking of dorsal horn alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits GluR1 and GluR2 under persistent inflammatory pain conditions and discusses the potential mechanisms by which such changes participate in the development and maintenance of inflammatory pain.

Chemokine signaling and the management of neuropathic pain.

Chemokines and chemokine receptors are widely expressed in the nervous system, where they play roles in the regulation of stem cell migration, axonal path finding, and neurotransmission. Chemokine signaling is also of key importance in the regulation of neuroinflammatory responses. The expression of the chemokine monocyte chemoattractant protein 1 (MCP1) and its receptor (CCR2) is upregulated by dorsal root ganglia neurons in rodent models of neuropathic pain. MCP1 increases the excitability of nociceptive neurons after a peripheral nerve injury, and disruption of MCP1 signaling blocks the development of neuropathic pain. In the spinal cord, microglial cells expressing CCR2 are thought to play an active role in the initiation and maintenance of pain hypersensitivity, and MCP1 may also alter the excitability of spinal neurons in some cases. Other predominant sites of CCR2 activation are found in the peripheral nervous system, thereby explaining, at least in some circumstances, the rapid anti-nociceptive effects of peripherally administered CCR2 antagonists. In this article we discuss the relative roles of CCR2 activation in the peripheral and central nervous systems in relation to the phenomenon of neuropathic pain.

Chemokine Network in the Nervous System: A New Target for Pain Relief

Physical insults including but not limited to nerve damage, inflammation, visceral pathologies and cancer generate long lasting pain commonly referred as chronic pain. Recently, members of the chemokine family and their receptors emerged as key modulators in nociceptive influx transmission in neuropathic and inflammatory chronic pain models. To this day, rodents defective in specific chemokine receptors have provided evidence of the implication of chemokine in pain sensitivity. In addition, up-regulation of chemokines and their receptors at multiple levels in the central nervous (CNS) and peripheral (PNS) systems is associated in the development of chronic pain. Indeed, we point out the fact that chemokines are synthesized and released by both neuronal and non-neuronal cells and act as neuromodulators. Even if their functional roles in the CNS remain largely unknown, chemokines participate in the glial activation and modulation of neuronal excitability as well as neurotransmitter release. This review focuses on three chemokines (i.e. CCL2, CXCL12, CX3CL1) recently identified as important mediators of the initiation and maintenance of pain hypersensitivity, thus broadening the panel of new strategies for the management of chronic pain.

Expression of CCR2 in Both Resident and Bone Marrow-Derived Microglia Plays a Critical Role in Neuropathic Pain

Neuropathic pain resulting from damage to or dysfunction of peripheral nerves is not well understood and difficult to treat. Although CNS hyperexcitability is a critical component, recent findings challenge the neuron-centric view of neuropathic pain etiology and pathology. Indeed, glial cells were shown to play an active role in the initiation and maintenance of pain hypersensitivity. However, the origins of these cells and the triggers that induce their activation have yet to be elucidated. Here we show that, after peripheral nerve injury induced by a partial ligation on the sciatic nerve, in addition to activation of microglia resident to the CNS, hematogenous macrophage/monocyte infiltrate the spinal cord, proliferate, and differentiate into microglia. Signaling from chemokine monocyte chemoattractant protein-1 (MCP-1, CCL2) to its receptor CCR2 is critical in the spinal microglial activation. Indeed, intrathecal injection of MCP-1 caused activation of microglia in wild-type but not in CCR2-deficient mice. Furthermore, treatment with an MCP-1 neutralizing antibody prevented bone marrow-derived microglia (BMDM) infiltration into the spinal cord after nerve injury. In addition, using selective knock-out of CCR2 in resident microglia or BMDM, we found that, although total CCR2 knock-out mice did not develop microglial activation or mechanical allodynia, CCR2 expression in either resident microglia or BMDM is sufficient for the development of mechanical allodynia. Thus, to effectively relieve neuropathic pain, both CNS resident microglia and blood-borne macrophages need to be targeted. These findings also open the door for a novel therapeutic strategy: to take advantage of the natural ability of bone marrow-derived cells to infiltrate selectively affected CNS regions by using these cells as vehicle for targeted drug delivery to inhibit hypersensitivity and chronic pain.

Polyamine deficient diet to relieve pain hypersensitivity

There is a compelling body of evidence that N-methyl-d-aspartate receptors (NMDA-R) play a critical role in the development and maintenance of pain hypersensitivity. However, long-term treatments with NMDA-R antagonists are limited by unacceptable side effects. Since polyamines modulate the functioning of NMDA-R and mainly originate from normal dietary intake and bacterial metabolism in the gut, we developed a nutritional therapy based on dietary polyamine deficiency. Here, we reported that a polyamine deficient diet (PD diet) for 7 days prevented the enhancement of tyrosine phosphorylation of the spinal NR2B subunit-containing NMDA-R associated with inflammation in rats. Based on these data, we studied the ability of PD diet to prevent long-lasting pain hypersensitivity associated with tissue injury on one hind paw by evaluating long-lasting changes in both mechanical nociceptive threshold and weight bearing. A PD diet strongly reduced long-lasting hyperalgesia induced by inflammation or incision, especially in fentanyl-treated rats. Moreover a PD diet also prevented the exaggerated hyperalgesia induced by a second inflammation performed 7 days after the first one. A PD diet also opposed paradoxical hyperalgesia induced by non-nociceptive environmental stress in rats with pain and opioid experiences. A PD diet reversed pain hypersensitivity associated with monoarthritis or neuropathy and restored the analgesic effect of morphine. Since PD diet was devoid of any noticeable side effects, this nutritional therapy could be part of an effective and safe strategy for pre-emptive analgesia and for reducing the transition from acute to chronic pain and its outcomes in various pain syndromes.