The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour.

Phosphorylation of histone H3 at serine 10 (p-H3S10) is a marker of active gene transcription. Using cognitive models of neural plasticity, p-H3S10 was shown to be downstream of extracellular signal-regulated kinase (ERK) signalling in the hippocampus. In this study, we show that nociceptive signalling after peripheral formalin injection increased p-H3S10 expression in the ipsilateral dorsal horn. This increase was maximal 30 minutes after formalin injection and occurred mainly within p-ERK-positive neurons. Spinal p-H3S10-enhanced expression was also observed in neurokinin 1 receptor (NK1R), c-Fos, and Zif268 positive neurons and was inhibited by ablation of serotonergic descending controls. The mitogen and stress-activated protein kinase 1 (MSK1) is downstream of ERK and can induce p-H3S10. We found that, after formalin injection, most phospho-MSK1 (p-MSK1)-positive cells (87% ± 3%) expressed p-ERK and the majority of p-H3S10-positive cells (85% ± 5%) expressed p-MSK1. Inhibition of ERK activity with the MEK inhibitor SL327 reduced formalin-induced p-ERK, p-MSK1, and p-H3S10, demonstrating that spinal p-MSK1 and p-H3S10 were at least partly downstream of ERK signalling. Crucially, pharmacological blockade of spinal MSK1 activity with the novel MSK1 inhibitor SB727651A inhibited formalin-induced spinal p-H3S10 and nocifensive behaviour. These findings are the first to establish the involvement of p-H3S10 and its main kinase, MSK1, in ERK regulation of nociception. Given the general importance of ERK signalling in pain processing, our results suggest that p-H3S10 could play a role in the response to injury.

Complex regulation of the regulator of synaptic plasticity histone deacetylase 2 in the rodent dorsal horn after peripheral injury.

Histone deacetylases (HDACs), HDAC2 in particular, have been shown to regulate various forms of learning and memory. Since cognitive processes share mechanisms with spinal nociceptive signalling, we decided to investigate the HDAC2 expression in the dorsal horn after peripheral injury. Using immunohistochemistry, we found that spinal HDAC2 was mainly seen in neurons and astrocytes, with neuronal expression in naïve tissue 2.6 times greater than that in astrocytes. Cysteine (S)-nitrosylation of HDAC2 releases HDAC2 gene silencing and is controlled by nitric oxide (NO). A duration of 48 h after intraplantar injection of complete Freund's adjuvant, there was an ipsilateral increase in the most important NO-producing enzyme in pain states, nitric oxide synthase (nNOS), accompanied by an increase in HDAC2 S-nitrosylation. Moreover, a subset of nNOS-positive neurons expressed cFos, a known target of HDAC2, suggesting that derepression of cFos expression following HDAC2 S-nitrosylation might occur after noxious stimulation. We saw no change in global HDAC2 expression in both short- and long-term pain states. However, HDAC2 was increased in astrocytes 7 days after neuropathic injury suggesting that HDAC2 might inhibit astrocytic gene expression in neuropathic pain states. All together, our results indicate that the epigenetic regulation of transcriptional programmes in the dorsal horn after injury is cell specific. Moreover, the prominent role of NO in persistent pain states suggests that HDAC2 S-nitrosylation could play a crucial role in the regulation of gene expression leading to hypersensitivity. Our manuscript describes for the first time the regulation of the memory regulator histone deacetylase 2 (HDAC2) in the superficial dorsal horn of adult rats following peripheral injury. Our cell-specific approach has revealed a complex pattern of expression of spinal HDAC2 that depends on the injury and the cell type, suggesting a sophisticated regulation of gene expression by HDAC2.

The stress regulator FKBP51 drives chronic pain by modulating spinal glucocorticoid signaling.

Polymorphisms in FKBP51 are associated with stress-related psychiatric disorders and influence the severity of pain symptoms experienced after trauma. We report that FKBP51 (FK506 binding protein 51) is crucial for the full development and maintenance of long-term pain states. Indeed, FKBP51 knockout mice, as well as mice in which silencing of FKBP51 is restricted to the spinal cord, showed reduced hypersensitivity in several persistent pain models in rodents. FKBP51 deletion did not compromise the detection of acute painful stimuli, a critical protective mechanism. Moreover, the intrathecal administration of the specific FKBP51 inhibitor SAFit2 reduced the severity of an established pain state, confirming the crucial role of spinal FKBP51 in nociceptive processing. Finally, glucocorticoid signaling, which is known to modulate persistent pain states in rodents, was impaired in FKBP51 knockout mice. This finding suggested that FKBP51 regulates chronic pain by modulation of glucocorticoid signaling. Thus, FKBP51 is a central mediator of chronic pain, likely in humans as well as rodents, and is a new pharmacologically tractable target for the treatment of long-term pain states.

Short-term anesthesia inhibits formalin-induced extracellular signal-regulated kinase (ERK) activation in the rostral anterior cingulate cortex but not in the spinal cord.

BACKGROUND: The rostral anterior cingulate cortex (rACC) has been implicated in the negative affective response to injury, and importantly, it has been shown that activation of extracellular signal-regulated kinase (ERK) signaling in the rACC contributes to the full expression of the affective component of pain in rodents. In this study, we investigated whether administration of anesthesia at the time of injury could reduce phosphorylated-ERK (PERK) expression in the rACC, which might eliminate the negative affective component of noxious stimulation. Intraplantar hindpaw formalin stimulation, an aversive event in the awake animal, was given with or without general isoflurane anesthesia, and PERK expression was subsequently quantified in the rACC using immunohistochemistry. Furthermore, as numerous studies have demonstrated the importance of spinal ERK signaling in the regulation of nociceptive behaviour, we also examined PERK in the superficial dorsal horn of the spinal cord. FINDINGS: Formalin injection with and without short-term (<10 min) general isoflurane anesthesia induced the same level of PERK expression in spinal cord laminae I-II. However, PERK expression was significantly inhibited across all laminae of the rACC in animals anesthetized during formalin injection. The effect of anesthesia was such that levels of PERK were the same in formalin and sham treated anesthesized animals. CONCLUSIONS: This study is the first to demonstrate that isoflurane anesthesia can inhibit formalin-induced PERK in the rACC and therefore might eliminate the unpleasantness of restraint associated with awake hindpaw injection.

Could targeting epigenetic processes relieve chronic pain states?

PURPOSE OF REVIEW: Aberrations in the epigenetic landscape have previously been associated with human diseases such as cancer and schizophrenia, and drugs that target epigenetic processes are currently used as therapeutic agents. This article will review the evidence obtained from animal studies indicating that epigenetic processes might regulate long-term pain states and then discuss the possibility that targeting epigenetic mechanisms might be useful for the management of chronic pain. RECENT FINDINGS: Recent animal studies have reported injury-induced changes in epigenetic processes in the central nervous system. The picture that has emerged is that of very complex epigenetic programs that depend on the injury. However, some studies have reported the successful use of nonspecific epigenetic tools to improve the hypersensitivity that develops in animal models of long-term pain states. SUMMARY: The field of epigenetics and pain is rapidly emerging but further investigation is needed to fully comprehend the contribution of epigenetic processes to chronic pain states. Although therapeutic approaches targeting these mechanisms might seem worthwhile, we cannot assert that currently available global tools such as histone deacetylase inhibitors can be used successfully for the long-term treatment of chronic pain states.

Regulation of gene expression and pain states by epigenetic mechanisms.

The induction of inflammatory or neuropathic pain states is known to involve molecular activity in the spinal superficial dorsal horn and dorsal root ganglia, including intracellular signaling events which lead to changes in gene expression. These changes ultimately cause alterations in macromolecular synthesis, synaptic transmission, and structural architecture which support central sensitization, a process required for the establishment of long-term pain states. Epigenetic mechanisms are essential for long-term synaptic plasticity and modulation of gene expression. This is because epigenetic modifications are known to regulate gene transcription by aiding the physical relaxation or condensation of chromatin. These processes are therefore potential regulators of the molecular changes underlying permanent pain states. A handful of studies have emerged in the field of pain epigenetics; however, the field is still very much in its infancy. This chapter draws upon other specialities which have extensively investigated epigenetic mechanisms, such as learning and memory and oncology. After defining epigenetics as well as the recent field of "neuroepigenetics" and the main molecular mechanisms involved, this chapter describes the role of these mechanisms in the synaptic plasticity seen in learning and memory, and address those epigenetic mechanisms that have been linked with the development of acute and prolonged pain states. Finally, the idea that long-lasting epigenetic modifications could contribute to the transition from acute to chronic pain states by supporting maladaptive molecular changes is discussed.

C-fiber activity-dependent maturation of glycinergic inhibition in the spinal dorsal horn of the postnatal rat.

Sensory circuits are shaped by experience in early postnatal life and in many brain areas late maturation of inhibition drives activity-dependent development. In the newborn spinal dorsal horn, activity is dominated by inputs from low threshold A fibers, whereas nociceptive C-fiber inputs mature gradually over the first postnatal weeks. How this changing afferent input influences the maturation of dorsal horn inhibition is not known. We show an absence of functional glycinergic inhibition in newborn dorsal horn circuits: Dorsal horn receptive fields and afferent-evoked excitation are initially facilitated by glycinergic activity due, at least in part, to glycinergic disinhibition of GAD67 cells. Glycinergic inhibitory control emerges in the second postnatal week, coinciding with an expression switch from neonatal α(2) homomeric to predominantly mature α(1)/ß glycine receptors (GlyRs). We further show that the onset of glycinergic inhibition depends upon the maturation of C-fiber inputs to the dorsal horn: selective block of afferent C fibers in postnatal week 2, using perisciatic injections of the cationic anesthetic QX-314, lidocaine, and capsaicin, delays the maturation of both GlyR subunits and glycinergic inhibition, maintaining dorsal neurons in a neonatal state, where tactile responses are facilitated, rather than inhibited, by glycinergic network activity. Thus, glycine may serve to facilitate tactile A-fiber-mediated information and enhance activity-dependent synaptic strengthening in the immature dorsal horn. This period ceases in the second postnatal week with the maturation of C-fiber spinal input, which triggers postsynaptic changes leading to glycinergic inhibition and only then is balanced excitation and inhibition achieved in dorsal horn sensory circuits.

The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states.

BACKGROUND: DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2) and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA) in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated. RESULTS: Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury. CONCLUSION: Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

Systemic inhibition of the mammalian target of rapamycin (mTOR) pathway reduces neuropathic pain in mice.

The management of neuropathic pain is unsatisfactory, and new treatments are required. Because the sensitivity of a subset of fast-conducting primary afferent nociceptors is thought to be regulated by the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, selectively targeting mTORC1 represents a new strategy for the control of chronic pain. Here we show that activated mTOR was expressed largely in myelinated sensory fibers in mouse and that inhibiting the mTORC1 pathway systemically alleviated mechanical hypersensitivity in mouse models of inflammatory and neuropathic pain. Specifically, systemic administration of mTORC1 inhibitor temsirolimus (CCI-779), both acutely (25 mg/kg i.p.) and chronically (4 daily 25 mg/kg i.p.), inhibited the mTORC1 pathway in sensory axons and the spinal dorsal horn and reduced mechanical and cold hypersensitivity induced by nerve injury. Moreover, systemic treatment with CCI-779 also reduced mechanical but not heat hypersensitivity in an inflammatory pain state. This treatment did not influence nociceptive thresholds in naive or sham-treated control animals. Also, there was no evidence for neuronal toxicity after repeated systemic treatment with CCI-779. Additionally, we show that acute and chronic i.p. administration of Torin1 (20 mg/kg), a novel ATP-competitive inhibitor targeting both mTORC1 and mTORC2 pathways, reduced the response to mechanical and cold stimuli in neuropathic mice. Our findings emphasize the importance of the mTORC1 pathway as a regulator of nociceptor sensitivity and therefore as a potential target for therapeutic intervention, particularly in chronic pain.

Injury induced activation of extracellular signal-regulated kinase (ERK) in the rat rostral ventromedial medulla (RVM) is age dependant and requires the lamina I projection pathway.

Descending controls originating in part from the rostral ventromedial medulla (RVM) regulate the excitability of dorsal horn neurons and maintain peripheral pain states. Activation of extracellular signal regulated kinase (ERK) in RVM neurons has been shown following peripheral inflammation and is involved in generating the accompanying inflammatory hyperalgesia. Here, we show that spared nerve injury (SNI), a model of neuropathic pain, results in an increase in ERK activity in RVM neurons of adult rats 3 and 8 days following surgery. We carried out two experimental procedures to demonstrate that this increase in ERK activation was related to the increased mechanical sensitivity associated with SNI. First, we showed that lesions of the lamina I/III ascending pathway from the dorsal horn attenuated both mechanical hyperalgesia and ERK activation in the RVM. Second, we performed SNI in P10 rats. At this age, SNI did not result in mechanical hypersensitivity, as previously shown, and did not activate ERK in the RVM. Finally, the percentage of pERK expressing neurones that were also serotonergic was always around 60%, independent of pain state and age, indicating an important role for serotonin in descending controls of pain states.

A rapamycin-sensitive signaling pathway is essential for the full expression of persistent pain states.

Translational control through the mammalian target of rapamycin (mTOR) is critical for synaptic plasticity, cell growth, and axon guidance. Recently, it was also shown that mTOR signaling was essential for the maintenance of the sensitivity of subsets of adult sensory neurons. Here, we show that persistent pain states, but not acute pain behavior, are substantially alleviated by centrally administered rapamycin, an inhibitor of the mTOR pathway. We demonstrate that rapamycin modulates nociception by acting on subsets of primary afferents and superficial dorsal horn neurons to reduce both primary afferent sensitivity and central plasticity. We found that the active form of mTOR is present in a subpopulation of myelinated dorsal root axons, but rarely in unmyelinated C-fibers, and heavily expressed in the dorsal horn by lamina I/III projection neurons that are known to mediate the induction and maintenance of pain states. Intrathecal injections of rapamycin inhibited the activation of downstream targets of mTOR in dorsal horn and dorsal roots and reduced the thermal sensitivity of A-fibers. Moreover, in vitro studies showed that rapamycin increased the electrical activation threshold of Adelta-fibers in dorsal roots. Together, our results imply that central rapamycin reduces neuropathic pain by acting both on an mTOR-positive subset of A-nociceptors and lamina I projection neurons and suggest a new pharmacological route for therapeutic intervention in persistent pain states.

Hindpaw incision in early life increases the hyperalgesic response to repeat surgical injury: critical period and dependence on initial afferent activity.

Pain in early life can enhance the response to subsequent injury, but effects are influenced by both the nature and timing of neonatal injury. Using plantar hindpaw incision, we investigated how postnatal age influences the response to repeat surgical injury two weeks later. The degree and time course of behavioural changes in mechanical withdrawal threshold were measured, and injury-related hyperalgesia was further quantified by flexion reflex electromyographic responses to suprathreshold mechanical stimuli 24 h following incision. Plantar hindpaw incision produces acute mechanical hyperalgesia in neonatal and adult rats, but incision in neonatal pups has an additional effect on the response to subsequent injury. With initial incision at postnatal day (P) 3 or 6, the degree of hyperalgesia following repeat incision 2 weeks later was greater than in animals having a single incision at the same age. At older ages (initial incision at P10, P21 or P40) responses did not differ in repeat and single incision groups. To test the role of primary afferent activity, levobupivacaine sciatic block was performed prior to P6 plantar incision, and controls received saline or subcutaneous levobupivacaine. Repeat peri-operative, but not a single pre-operative sciatic block, prevented the enhanced response to repeat incision two weeks later. Our results show that the first postnatal week represents a critical period when incision increases hyperalgesia following repeat surgery two weeks later, and effects are initiated by peripheral afferent activity. This has potential therapeutic implications for the type and duration of peri-operative analgesia used for neonatal surgery.

Descending serotonergic controls regulate inflammation-induced mechanical sensitivity and methyl-CpG-binding protein 2 phosphorylation in the rat superficial dorsal horn.

BACKGROUND: Regulation of pain states is, in part, dependent upon plastic changes in neurones within the superficial dorsal horn. There is also compelling evidence that pain states are under the control of descending projections from the brainstem. While a number of transcription factors including Methyl-CpG-binding protein 2 (MeCP2), Zif268 and Fos have been implicated in the regulation of dorsal horn neurone sensitization following injury, modulation of their activity by descending controls has not been investigated. RESULTS: Here, we describe how descending controls regulate MeCP2 phosphorylation (P-MeCP2), known to relieve transcriptional repression by MeCP2, and Zif268 and Fos expression in the rat superficial dorsal horn, after CFA injection into the hind paw. First, we report that CFA significantly increased P-MeCP2 in Lamina I and II, from 30 min post injection, with a maximum reached after 1 h. The increase in P-MeCP2 paralleled that of Zif268 and Fos, and P-MeCP2 was expressed in large sub-populations of Zif268 and Fos expressing neurones. Serotonergic depletion of the lumbar spinal cord with 5,7 di-hydroxytryptamine creatinine sulphate (5,7-DHT) reduced the inflammation evoked P-MeCP2 in the superficial dorsal horn by 57%, and that of Zif268 and Fos by 37.5% and 30% respectively. Although 5,7-DHT did not change primary thermal hyperalgesia, it significantly attenuated mechanical sensitivity seen in the first 24 h after CFA. CONCLUSION: We conclude that descending serotonergic pathways play a crucial role in regulating gene expression in the dorsal horn and mechanical sensitivity associated with an inflammatory pain state.