Intrathecal delivery of histone deacetylase inhibitors ameliorates hypersensitivity in models of neuropathic pain. This effect may be mediated at the level of the spinal cord through inhibition of HDAC1 function.
Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug–induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.
Histone deacetylase; Histone deacetylase inhibitors; Neuropathic pain
Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.
Neuregulin 1 acts as an axonal signal that regulates multiple aspects of Schwann cell
development including the survival and migration of Schwann cell precursors, the
ensheathment of axons and subsequent elaboration of the myelin sheath. To examine the role
of this factor in remyelination and repair following nerve injury, we ablated neuregulin 1
in the adult nervous system using a tamoxifen inducible Cre recombinase transgenic mouse
system. The loss of neuregulin 1 impaired remyelination after nerve crush, but did not
affect Schwann cell proliferation associated with Wallerian degeneration or axon
regeneration or the clearance of myelin debris by macrophages. Myelination changes were
most marked at 10 days after injury but still apparent at 2 months post-crush.
Transcriptional analysis demonstrated reduced expression of myelin-related genes during
nerve repair in animals lacking neuregulin 1. We also studied repair over a prolonged time
course in a more severe injury model, sciatic nerve transection and reanastamosis. In the
neuregulin 1 mutant mice, remyelination was again impaired 2 months after nerve
transection and reanastamosis. However, by 3 months post-injury axons lacking neuregulin 1
were effectively remyelinated and virtually indistinguishable from control. Neuregulin 1
signalling is therefore an important factor in nerve repair regulating the rate of
remyelination and functional recovery at early phases following injury. In contrast to
development, however, the determination of myelination fate following nerve injury is not
dependent on axonal neuregulin 1 expression. In the early phase following injury, axonal
neuregulin 1 therefore promotes nerve repair, but at late stages other signalling pathways
appear to compensate.
injury; Nrg1; regeneration; remyelination; Schwann
In the majority of spinal cord injuries (SCIs) some axonal projections remain intact. We examined the functional status of these surviving axons, since they represent a prime therapeutic target. Using a novel electrophysiological preparation, adapted from techniques used to study primary demyelination, we quantified conduction failure across a SCI and studied conduction changes over time in adult rats with a moderate severity spinal contusion (150 kilodyne, Infinite Horizon impactor). By recording antidromically activated single units from teased dorsal root filaments we demonstrate complete conduction block in ascending dorsal column axons acutely (1-7 days) post-injury, followed by a period of restored conduction over the sub-acute phase (2-4 weeks), with no further improvements in conduction at chronic stages (3-6 months). By cooling the lesion site additional conducting fibres could be recruited, thus revealing a population of axons that are viable but unable to conduct under normal physiological conditions. Importantly, this phenomenon is still apparent at the most chronic (6 month) time point. The time course of conduction changes corresponded with changes in behavioural function, and ultrastructural analysis of dorsal column axons revealed extensive demyelination during the period of conduction block, followed by progressive remyelination. A proportion of dorsal column axons remained chronically demyelinated, suggesting that these are the axons recruited with the cooling paradigm. Thus, using a clinically relevant SCI model we have identified a population of axons present at chronic injury stages which are intact but fail to conduct and are therefore a prime target for therapeutic strategies to restore function.
Chondroitinase ABC (ChABC) represents a promising therapeutic strategy for the treatment of spinal cord injury due to its potent effects on restoring function to spinal injured adult mammals. However, there is limited mechanistic insight as to the underlying effects of ChABC treatment, where the effects are mediated, and which signalling pathways are involved in ChABC-mediated repair. Here we utilise a transgenic (YFP-H) mouse to demonstrate that cortical layer V projection neurons undergo severe atrophy four weeks following thoracic dorsal column injury and that ChABC is neuroprotective for these neurons following ICV infusion. ChABC also prevented cell atrophy following localised delivery to the spinal cord, suggesting a possible retrograde neuroprotective effect mediated at the injury site. Furthermore, neuroprotection of corticospinal cell somata coincided with increased axonal sprouting in the spinal cord. In addition, Western blot analysis of a number of kinases important in survival and growth signalling revealed a significant increase in phosphorylated ERK1 at the spinal injury site following in vivo ChABC treatment, indicating that activated ERK may play a role in downstream repair processes following ChABC treatment. Total forms of PKC and AKT were also elevated, indicating that modification of the glial scar by ChABC promotes long-lasting signalling changes at the lesion site. Thus, using the YFP-H mouse as a novel tool to study degenerative changes and repair following spinal cord injury we demonstrate, for the first time, that ChABC treatment regulates multiple signalling cascades at the injury site and exerts protective effects on axotomised corticospinal projection neurons.
spinal cord injury; proteoglycan; neuroprotection; regeneration; repair; transgenic
Small proline-rich repeat protein 1A (SPRR1A) is expressed in dorsal root ganglion (DRG) neurons following peripheral nerve injury but it is not known whether SPRR1A is differentially expressed following injury to peripheral versus central DRG projections and a detailed characterisation of expression in sensory neuron sub-populations and spinal cord has not been performed. Here we use immunocytochemical techniques to characterise SPRR1A expression following sciatic nerve, dorsal root and dorsal column injury in adult mice. SPRR1A was not detected in naïve spinal cord, DRG or peripheral nerves and there was minimal expression following injury to the centrally projecting branches of DRG neurons. However, following peripheral (sciatic) nerve injury, intense SPRR1A immunoreactivity was observed in the dorsal horn and motoneurons of the spinal cord, in L4/5 DRG neurons and in the injured nerve. A time-course study comparing expression following sciatic nerve crush and transection revealed maximum SPRR1A levels at day 7 in both models. However, while SPRR1A was down-regulated to baseline by 30 days post-lesion following crush injury, it remained elevated 30 days after transection. Cell-size and double-labelling studies revealed that SPRR1A was expressed by DRG cells of all sizes and co-localized with classical markers of DRG subpopulations and their primary afferent terminals. High co-expression of SPRR1A with activating transcription factor-3 and growth-associated protein-43 was observed, indicating that it is expressed by injured and regenerating neurons. This study supports the hypothesis that SPRR1A is a regeneration-associated gene and that SPRR1A provides a valuable marker to assess the regenerative potential of injured neurons.
nerve injury and repair; axonal regeneration; immunolabelling; primary afferents; dorsal root ganglia; regeneration-associated genes
Many persistent pain states (pain lasting for hours, days, or longer) are poorly treated because of the limitations of existing therapies. Analgesics such as nonsteroidal anti-inflammatory drugs and opioids often provide incomplete pain relief and prolonged use results in the development of severe side effects. Identification of the key mediators of various types of pain could improve such therapies. Here, we tested the hypothesis that hitherto unrecognized cytokines and chemokines might act as mediators in inflammatory pain. We used ultraviolet B (UVB) irradiation to induce persistent, abnormal sensitivity to pain in humans and rats. The expression of more than 90 different inflammatory mediators was measured in treated skin at the peak of UVB-induced hypersensitivity with custom-made polymerase chain reaction arrays. There was a significant positive correlation in the overall expression profiles between the two species. The expression of several genes [interleukin-1β (IL-1β), IL-6, and cyclooxygenase-2 (COX-2)], previously shown to contribute to pain hypersensitivity, was significantly increased after UVB exposure, and there was dysregulation of several chemokines (CCL2, CCL3, CCL4, CCL7, CCL11, CXCL1, CXCL2, CXCL4, CXCL7, and CXCL8). Among the genes measured, CXCL5 was induced to the greatest extent by UVB treatment in human skin; when injected into the skin of rats, CXCL5 recapitulated the mechanical hypersensitivity caused by UVB irradiation. This hypersensitivity was associated with the infiltration of neutrophils and macrophages into the dermis, and neutralizing the effects of CXCL5 attenuated the abnormal pain-like behavior. Our findings demonstrate that the chemokine CXCL5 is a peripheral mediator of UVB-induced inflammatory pain, likely in humans as well as rats.
Peripheral inflammation or nerve injury induces a primary afferent barrage into the spinal cord, which can cause N-methyl -aspartate (NMDA) receptor-dependent alterations in the responses of dorsal horn sensory neurons to subsequent afferent inputs. This plasticity, such as “wind-up” and central sensitization, contributes to the hyperexcitability of dorsal horn neurons and increased pain-related behavior in animal models, as well as clinical signs of chronic pain in humans, hyperalgesia and allodynia. Binding of NMDA receptor subunits by the scaffolding protein postsynaptic density protein-95 (PSD-95) can facilitate downstream intracellular signaling and modulate receptor stability, contributing to synaptic plasticity. Here, we show that spinal delivery of the mimetic peptide Tat-NR2B9c disrupts the interaction between PSD-95 and NR2B subunits in the dorsal horn and selectively reduces NMDA receptor-dependent events including wind-up of spinal sensory neurons, and both persistent formalin-induced neuronal activity and pain-related behaviors, attributed to central sensitization. Furthermore, a single intrathecal injection of Tat-NR2B9c in rats with established nerve injury-induced pain attenuates behavioral signs of mechanical and cold hypersensitivity, with no effect on locomotor performance. Thus, uncoupling of PSD-95 from spinal NR2B-containing NMDA receptors may prevent the neuronal plasticity involved in chronic pain and may be a successful analgesic therapy, reducing side effects associated with receptor blockade.
A painful neuropathy is frequently observed in people living with human immunodeficiency virus type 1 (HIV-1). The HIV coat protein, glycoprotein 120 (gp120), implicated in the pathogenesis of neurological disorders associated with HIV, is capable of initiating neurotoxic cascades via an interaction with the CXCR4 and/or CCR5 chemokine receptors, which may underlie the pathogenesis of HIV-associated peripheral neuropathic pain. In order to elucidate the mechanisms underlying HIV-induced painful peripheral neuropathy, we have characterised pathological events in the peripheral and central nervous system following application of HIV-1 gp120 to the rat sciatic nerve. Perineural HIV-1 gp120 treatment induced a persistent mechanical hypersensitivity (44% decrease from baseline), but no alterations in sensitivity to thermal or cold stimuli, and thigmotactic (anxiety-like) behaviour in the open field. The mechanical hypersensitivity was sensitive to systemic treatment with gabapentin, morphine and the cannabinoid WIN 55,212-2, but not with amitriptyline. Immunohistochemical studies reveal: decreased intraepidermal nerve fibre density, macrophage infiltration into the peripheral nerve at the site of perineural HIV-1 gp120; changes in sensory neuron phenotype including expression of activating transcription factor 3 (ATF3) in 27% of cells, caspase-3 in 25% of cells, neuropeptide Y (NPY) in 12% of cells and galanin in 13% of cells and a spinal gliosis. These novel findings suggest that this model is not only useful for the elucidation of mechanisms underlying HIV-1-related peripheral neuropathy but may prove useful for preclinical assessment of drugs for the treatment of HIV-1 related peripheral neuropathic pain.
HIV-1; Neuropathy; Microglia; Macrophages; Gabapentin; Cannabinoids
P2X3 and P2X2/3 receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X3 and P2X2/3 antagonist, AF-792 (previously known as RO-5), in addition to less selective purinoceptor ligands, was applied intrathecally in vivo. Cystometry recordings were made to assess changes in the micturition reflex contractions following drug treatments. We found AF-792 inhibited micturition reflex activity significantly (300 nmol; from baseline contraction intervals of 1.18 ± 0.07 min to 9.33 ± 2.50 min). Furthermore, inhibition of P2X3 and P2X2/3 receptors in the spinal cord significantly attenuated spinal activation of extracellular-signal regulated kinases (ERK) induced by acute peripheral stimulation of the bladder with 1% acetic acid by 46.4 ± 12.0 % on average. Hence, the data suggest that afferent signals originating from the bladder are regulated by spinal P2X3 and P2X2/3 receptors and establish directly an endogenous central presynaptic purinergic mechanism to regulate visceral sensory transmission. Identification of this spinal purinergic control in visceral activities may help the development of P2X3 and P2X2/3 antagonist to treat urological dysfunction such as overactive bladder and possibly other debilitating sensory disorders including chronic pain states.
P2X; spinal cord; presynaptic; extracellular-signal regulated kinases; bladder; afferent
Here we show that phosphatidylinositol 3-kinase (PI3K) is a key player in the establishment of central sensitization, the spinal cord phenomenon associated with persistent afferent inputs and contributing to chronic pain states. We demonstrated electrophysiologically that PI3K is required for the full expression of spinal neuronal wind-up. In an inflammatory pain model, intrathecal administration of LY294002, a potent PI3K inhibitor, dose-dependently inhibited pain related behavior. This effect was correlated with a reduction of the phosphorylation of extracellular signal-regulated kinase (ERK) and CaMKinase II. In addition, we observed a significant decrease in the phosphorylation of the NMDA receptor subunit NR2B, decreased translocation to the plasma membrane of the GluR1 AMPA receptor subunit in the spinal cord and a reduction of evoked neuronal activity as measured using c-Fos immunohistochemistry. Our study suggests that PI3K is a major factor in the expression of central sensitization after noxious inflammatory stimuli.
Phosphorylation; ERK; GluR1; CaMKII; NMDA; formalin
The cytokine Interleukin-1β (IL-1β) released by spinal microglia in enhanced response states contributes significantly to neuronal mechanisms of chronic pain. Here we examine the involvement of the purinergic P2X7 receptor in the release of IL-1β following activation of Toll-like receptor-4 (TLR4) in the dorsal horn, which is associated with nociceptive behaviour and microglial activation. We observed that LPS induced release of IL-1β was prevented by pharmacological inhibition of the P2X7 receptor with A-438079, and was absent in spinal cord slices taken from P2X7 knock-out mice. Application of ATP did not evoke release of IL-1β from the dorsal horn unless preceded by an LPS priming stimulus, and this release was dependent on P2X7 receptor activation. Extensive phosphorylation of p38 MAPK in microglial cells in the dorsal horn was found to correlate with IL-1β secretion following both LPS and ATP. In behavioural studies, intrathecal injection of LPS in the lumbar spinal cord produced mechanical hyperalgesia in rat hind-paws which was attenuated by concomitant injections of either a non-specific (oxidized ATP) or a specific (A-438079) P2X7 antagonist. In addition, LPS induced hypersensitivity was observed in wild-type, but not P2X7 knock-out mice. These data suggest a critical role for the P2X7 receptor in the enhanced nociceptive transmission associated with microglial activation and secretion of IL-1β in the dorsal horn. We suggest that CNS penetrant P2X7 receptor antagonists, by targeting microglia in pain-enhanced response states, may be beneficial for the treatment of persistent pain.
Neuroinflammation; Cytokines; Microglia; Release; Lipopolysaccharide; ATP
Overexpression of neuronal calcium sensor 1 in cortical neurons can help restore axonal plasticity and regeneration following axonal injury in adult rats, and can also improve behavioral function.
Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST) normally have low levels of the neuronal calcium sensor-1 (NCS1) protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system.
Following trauma to the central nervous system (brain or spinal cord), neurons show very little capacity to re-grow their axons, which can lead to a permanent loss of function in those regions. In this study, we show that this failure of axon re-growth is associated with low intracellular levels of a small molecule called neuronal calcium sensor-1 (NCS1). We modified a non-replicating virus in two ways so as to increase the level of NCS1 in neurons while simultaneously labelling them with a green fluorescent protein, which allowed us to track neuronal growth. Using this virus to increase the level of NCS1 in a particular group of neurons that communicate between the brain and spinal cord, we showed that new axonal growth occurred in the spinal cord with or without injury to the neurons. Electrophysiological assessments demonstrated that these new processes formed functional connections in the spinal cord, and behavioural experiments revealed that this recovery also helped the animals move their limbs more effectively. Furthermore, an increase in NCS1 protected these neurons, such that fewer of them died after the injury. These findings demonstrate that increasing the intracellular levels of NCS1 in neurons can aid in the recovery from central nervous system injury, and can help improve behavioural function.
To elucidate the mechanisms underlying peripheral neuropathic pain in the context of HIV infection and antiretroviral therapy, we measured gene expression in dorsal root ganglia (DRG) of rats subjected to systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) and concomitant delivery of HIV-gp120 to the rat sciatic nerve. L4 and L5 DRGs were collected at day 14 (time of peak behavioural change) and changes in gene expression were measured using Affymetrix whole genome rat arrays. Conventional analysis of this data set and Gene Set Enrichment Analysis (GSEA) was performed to discover biological processes altered in this model. Transcripts associated with G protein coupled receptor signalling and cell adhesion were enriched in the treated animals, while ribosomal proteins and proteasome pathways were associated with gene down-regulation. To identify genes that are directly relevant to neuropathic mechanical hypersensitivity, as opposed to epiphenomena associated with other aspects of the response to a sciatic nerve lesion, we compared the gp120 + ddC-evoked gene expression with that observed in a model of traumatic neuropathic pain (L5 spinal nerve transection), where hypersensitivity to a static mechanical stimulus is also observed. We identified 39 genes/expressed sequence tags that are differentially expressed in the same direction in both models. Most of these have not previously been implicated in mechanical hypersensitivity and may represent novel targets for therapeutic intervention. As an external control, the RNA expression of three genes was examined by RT-PCR, while the protein levels of two were studied using western blot analysis.
Neuropathic pain; HIV; Mechanical hypersensitivity; Microarray
A distal symmetrical sensory peripheral neuropathy is frequently observed in people living with Human Immunodeficiency Virus Type 1 (HIV-1). This neuropathy can be associated with viral infection alone, probably involving a role for the envelope glycoprotein gp120; or a drug-induced toxic neuropathy associated with the use of nucleoside analogue reverse transcriptase inhibitors as a component of highly active anti-retroviral therapy. In order to elucidate the mechanisms underlying drug-induced neuropathy in the context of HIV infection, we have characterized pathological events in the peripheral and central nervous system following systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) with or without the concomitant delivery of HIV-gp120 to the rat sciatic nerve (gp120+ddC). Systemic ddC treatment alone is associated with a persistent mechanical hypersensitivity (33% decrease in limb withdrawal threshold) that when combined with perineural HIV-gp120 is exacerbated (48% decrease in threshold) and both treatments result in thigmotactic (anxiety-like) behaviour. Immunohistochemical studies revealed little ddC-associated alteration in DRG phenotype, as compared with known changes following perineural HIV-gp120. However, the chemokine CCL2 is significantly expressed in the DRG of rats treated with perineural HIV-gp120 and/or ddC and there is a reduction in intraepidermal nerve fibre density, comparable to that seen in herpes zoster infection. Moreover, a spinal gliosis is apparent at times of peak behavioural sensitivity that is exacerbated in gp120+ddC as compared to either treatment alone. Treatment with the microglial inhibitor, minocycline, is associated with delayed onset of hypersensitivity to mechanical stimuli in the gp120+ddC model and reversal of some measures of thigmotaxis. Finally, the hypersensitivity to mechanical stimuli was sensitive to systemic treatment with gabapentin, morphine and the cannabinoid WIN 55,212-2, but not with amitriptyline. These data suggests that both neuropathic pain models display many features of HIV- and anti-retroviral-related peripheral neuropathy. They therefore merit further investigation for the elucidation of underlying mechanisms and may prove useful for preclinical assessment of drugs for the treatment of HIV-related peripheral neuropathic pain.
HIV; anti-retroviral drugs; neuropathy; DRG; microglia
Changes in sodium channel activity and neuronal hyperexcitability contribute to neuropathic pain, a major clinical problem. There is strong evidence that the re-expression of the embryonic voltage-gated sodium channel subunit Nav1.3 underlies neuronal hyperexcitability and neuropathic pain.
Here we show that acute and inflammatory pain behaviour is unchanged in global Nav1.3 mutant mice. Surprisingly, neuropathic pain also developed normally in the Nav1.3 mutant mouse. To rule out any genetic compensation mechanisms that may have masked the phenotype, we investigated neuropathic pain in two conditional Nav1.3 mutant mouse lines. We used Nav1.8-Cre mice to delete Nav1.3 in nociceptors at E14 and NFH-Cre mice to delete Nav1.3 throughout the nervous system postnatally. Again normal levels of neuropathic pain developed after nerve injury in both lines. Furthermore, ectopic discharges from damaged nerves were unaffected by the absence of Nav1.3 in global knock-out mice. Our data demonstrate that Nav1.3 is neither necessary nor sufficient for the development of nerve-injury related pain.
Sensitivity to pain varies considerably between individuals and is known to be heritable. Increased sensitivity to experimental pain is a risk factor for developing chronic pain, a common and debilitating but poorly understood symptom. To understand mechanisms underlying pain sensitivity and to search for rare gene variants (MAF<5%) influencing pain sensitivity, we explored the genetic variation in individuals' responses to experimental pain. Quantitative sensory testing to heat pain was performed in 2,500 volunteers from TwinsUK (TUK): exome sequencing to a depth of 70× was carried out on DNA from singletons at the high and low ends of the heat pain sensitivity distribution in two separate subsamples. Thus in TUK1, 101 pain-sensitive and 102 pain-insensitive were examined, while in TUK2 there were 114 and 96 individuals respectively. A combination of methods was used to test the association between rare variants and pain sensitivity, and the function of the genes identified was explored using network analysis. Using causal reasoning analysis on the genes with different patterns of SNVs by pain sensitivity status, we observed a significant enrichment of variants in genes of the angiotensin pathway (Bonferroni corrected p = 3.8×10−4). This pathway is already implicated in animal models and human studies of pain, supporting the notion that it may provide fruitful new targets in pain management. The approach of sequencing extreme exome variation in normal individuals has provided important insights into gene networks mediating pain sensitivity in humans and will be applicable to other common complex traits.
Chronic widespread pain is a complex clinical problem. Identification of underlying genetic factors would shed light on the biology of pain and offer targets for novel therapies. We aimed to identify rare genetic variants in the normal population associated with pain sensation by performing exome sequencing on individuals who were more or less sensitive to heat pain. While we did not identify any single variants having large effect, we did observe major group differences between the sensitive and insensitive individuals. Network analysis suggested a role for the angiotensin pathway, which previous work in animal models has suggested is important in pain mediation. Our results cast light on the genetic factors underlying normal pain sensation in humans and the utility of exome analyses. It suggests that further exploration of the angiotensin pathway may reveal novel targets for the treatment of pain.
Central sensitization requires the activation of various intracellular signalling pathways within spinal dorsal horn neurons, leading to a lowering of activation threshold and enhanced responsiveness of these cells. Such plasticity contributes to the manifestation of chronic pain states and displays a number of features of long-term potentiation (LTP), a ubiquitous neuronal mechanism of increased synaptic strength. Here we describe the role of a novel pathway involving atypical PKCζ/PKMζ in persistent spinal nociceptive processing, previously implicated in the maintenance of late-phase LTP.
Using both behavioral tests and in vivo electrophysiology in rats, we show that inhibition of this pathway, via spinal delivery of a myristoylated protein kinase C-ζ pseudo-substrate inhibitor, reduces both pain-related behaviors and the activity of deep dorsal horn wide dynamic range neurons (WDRs) following formalin administration. In addition, Complete Freund's Adjuvant (CFA)-induced mechanical and thermal hypersensitivity was also reduced by inhibition of PKCζ/PKMζ activity. Importantly, this inhibition did not affect acute pain or locomotor behavior in normal rats and interestingly, did not inhibited mechanical allodynia and hyperalgesia in neuropathic rats. Pain-related behaviors in both inflammatory models coincided with increased phosphorylation of PKCζ/PKMζ in dorsal horn neurons, specifically PKMζ phosphorylation in formalin rats. Finally, inhibition of PKCζ/PKMζ activity decreased the expression of Fos in response to formalin and CFA in both superficial and deep laminae of the dorsal horn.
These results suggest that PKCζ, especially PKMζ isoform, is a significant factor involved in spinal persistent nociceptive processing, specifically, the manifestation of chronic pain states following peripheral inflammation.
atypical PKCζ; persistent spinal nociceptive processing; inflammatory pain; dorsal horn; Fos