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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

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

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 DNA methylation signature in the stress driver gene Fkbp5 indicates a neuropathic component in chronic pain

BACKGROUND: Epigenetic changes can bring insight into gene regulatory mechanisms associated with disease pathogenicity, including chronicity and increased vulnerability. To date, we are yet to identify genes sensitive to epigenetic regulation that contribute to the maintenance of chronic pain and with an epigenetic landscape indicative of the susceptibility to persistent pain. Such genes would provide a novel opportunity for better pain management, as their epigenetic profile could be targeted for the treatment of chronic pain or used as an indication of vulnerability for prevention strategies. Here, we investigated the epigenetic profile of the gene Fkbp5 for this potential, using targeted bisulphite sequencing in rodent pre-clinical models of chronic and latent hypersensitive states. RESULTS: The Fkbp5 promoter DNA methylation (DNAm) signature in the CNS was significantly different between models of persistent pain, and there was a significant correlation between CNS and peripheral blood Fkbp5 DNAm, indicating that further exploration of Fkbp5 promoter DNAm as an indicator of chronic pain pathogenic origin is warranted. We also found that maternal separation, which promotes the persistency of inflammatory pain in adulthood, was accompanied by long-lasting reduction in Fkbp5 DNAm, suggesting that Fkbp5 DNAm profile may indicate the increased vulnerability to chronic pain in individuals exposed to trauma in early life. CONCLUSIONS: Overall, our data demonstrate that the Fkbp5 promoter DNAm landscape brings novel insight into the differing pathogenic origins of chronic pain, may be able to stratify patients and predict the susceptibility to chronic pain

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: a novel and promising druggable target for the treatment of persistent pain states across sexes

It is well established that FKBP51 regulates the stress system by modulating the sensitivity of the glucocorticoid receptor to stress hormones. Recently, we have demonstrated that FKBP51 also drives long-term inflammatory pain states in male mice by modulating glucocorticoid signalling at spinal cord level. Here, we explored the potential of FKBP51 as a new pharmacological target for the treatment of persistent pain across the sexes. First, we demonstrated that FKBP51 regulates long-term pain states of different aetiologies independently of sex. Deletion of FKBP51 reduced the mechanical hypersensitivity seen in joint inflammatory and neuropathic pain states in female and male mice. Furthermore, FKBP51 deletion also reduced the hypersensitivity seen in a translational model of chemotherapy-induced pain. Interestingly, these 3 pain states were associated with changes in glucocorticoid signalling, as indicated by the increased expression, at spinal cord level, of the glucocorticoid receptor isoform associated with glucocorticoid resistance, GRβ, and increased levels of plasma corticosterone. These pain states were also accompanied by an upregulation of interleukin-6 in the spinal cord. Crucially, we were able to pharmacologically reduce the severity of the mechanical hypersensitivity seen in these 3 models of persistent pain with the unique FKBP51 ligand SAFit2. When SAFit2 was combined with a state-of-the-art vesicular phospholipid gel formulation for slow release, a single injection of SAFit2 offered pain relief for at least 7 days. We therefore propose the pharmacological blockade of FKBP51 as a new approach for the treatment of persistent pain across sexes, likely in humans as well as rodents

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