Margarita Calvo

The role of the immune system in the generation of neuropathic pain

Persistent pain is a sequela of several neurological conditions with a primary immune basis, such as Guillain-Barré syndrome and multiple sclerosis. Additionally, diverse forms of injury to the peripheral or the central nervous systems--whether traumatic, metabolic, or toxic--result in substantial recruitment and activation of immune cells. This response involves the innate immune system, but evidence also exists of T-lymphocyte recruitment, and in some patient cohorts antibodies to neuronal antigens have been reported. Mediators released by immune cells, such as cytokines, sensitise nociceptive signalling in the peripheral and central nervous systems. Preclinical data suggest an immune pathogenesis of neuropathic pain, but clinical evidence of a central role of the immune system is less clear. An important challenge for the future is to establish to what extent this immune response initiates or maintains neuropathic pain in patients and thus whether it is amenable to therapy.

Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia

In neurodegenerative conditions and following brain trauma it is not understood why neurons die while astrocytes and microglia survive and adopt pro-inflammatory phenotypes. We show here that the damaged adult brain releases diffusible factors that can kill cortical neurons and we have identified histone H1 as a major extracellular candidate that causes neurotoxicity and activation of the innate immune system. Extracellular core histones H2A, H2B H3 and H4 were not neurotoxic. Innate immunity in the central nervous system is mediated through microglial cells and we show here for the first time that histone H1 promotes their survival, up-regulates MHC class II antigen expression and is a powerful microglial chemoattractant. We propose that when the central nervous system is degenerating, histone H1 drives a positive feedback loop that drives further degeneration and activation of immune defences which can themselves be damaging. We suggest that histone H1 acts as an antimicrobial peptide and kills neurons through mitochondrial damage and apoptosis.

Neuregulin-ErbB Signaling Promotes Microglial Proliferation and Chemotaxis Contributing to Microgliosis and Pain after Peripheral Nerve Injury

A key component in the response of the nervous system to injury is the proliferation and switch to a “proinflammatory” phenotype by microglia (microgliosis). In situations where the blood–brain barrier is intact, microglial numbers increase via the proliferation and chemotaxis of resident microglia; however, there is limited knowledge regarding the factors mediating this response. After peripheral nerve injury, a dorsal horn microgliosis develops, which directly contributes to the development of neuropathic pain. Neuregulin-1 (NRG-1) is a growth and differentiation factor with a well characterized role in neural and cardiac development. Microglia express the NRG1 receptors erbB2, 3, and 4, and NRG1 signaling via the erbB2 receptor stimulated microglial proliferation, chemotaxis, and survival, as well as interleukin-1β release in vitro. Intrathecal treatment with NRG1 resulted in microglial proliferation within the dorsal horn, and these cells developed an activated morphology. This microglial response was associated with the development of both mechanical and cold pain-related hypersensitivity. Primary afferents express NRG1, and after spinal nerve ligation (SNL) we observed both an increase in NRG1 within the dorsal horn as well as activation of erbB2 specifically within microglia. Blockade of the erbB2 receptor or sequestration of endogenous NRG after SNL reduced the proliferation, the number of microglia with an activated morphology, and the expression of phospho-P38 by microglia. Furthermore, consequent to such changes, the mechanical pain-related hypersensitivity and cold allodynia were reduced. NRG1-erbB signaling therefore represents a novel pathway regulating the injury response of microglia.

Following Nerve Injury Neuregulin-1 Drives Microglial Proliferation and Neuropathic Pain via the MEK/ERK Pathway

Following peripheral nerve injury microglia accumulate within the spinal cord and adopt a proinflammatory phenotype a process which contributes to the development of neuropathic pain. We have recently shown that neuregulin‐1, a growth factor released following nerve injury, activates erbB 2, 3, and 4 receptors on microglia and stimulates proliferation, survival and chemotaxis of these cells. Here we studied the intracellular signaling pathways downstream of neuregulin‐1‐erbB activation in microglial cells. We found that neuregulin‐1 in vitro induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. Using specific kinase inhibitors we found that the mitogenic effect of neuregulin‐1 on microglia was dependant on MEK/ERK1/2 pathway, the chemotactic effect was dependant on PI3K/Akt signaling and survival was dependant on both pathways. Intrathecal treatment with neuregulin‐1 was associated with microgliosis and development of mechanical and cold pain related hypersensitivity which was dependant on ERK1/2 phosphorylation in microglia. Spinal nerve ligation results in a robust microgliosis and sustained ERK1/2 phosphorylation within these cells. This pathway is downstream of neuregulin‐1/erbB signaling since its blockade resulted in a significant reduction in microglial ERK1/2 phosphorylation. Inhibition of the MEK/ERK1/2 pathway resulted in decreased spinal microgliosis and in reduced mechanical and cold hypersensitivity after peripheral nerve damage. We conclude that neuregulin‐1 released after nerve injury activates microglial erbB receptors which consequently stimulates the MEK/ERK1/2 pathway that drives microglial proliferation and contributes to the development of neuropathic pain.

CXCL5 mediates UVB irradiation-induced pain.

The cytokine CXCL5 is a peripheral mediator of pain induced by UVB irradiation to the skin. Pinpointing the Cause of Sunburns Pain As any summer sunbather knows, when pain persists after the immediate cause is removed, it can be debilitating and can cause delayed problems such as cancer. To better understand this undesirable form of pain, Dawes et al. examine sunburned skin, a good example of inflammatory pain. Pain caused by ultraviolet B (UVB) light–induced DNA damage, these investigators found, is caused by the cytokine CXCL5. They made sure that their results from ras apply to human skin by carefully comparing how both human and rodent skin react to UVB irradiation. UV irradiation of rat foot and human forearm skin causes increased blood flow and painful hypersensitivity to mechanical and heat-induced stimuli 40 hours later. By using a large array that detected expression of many cytokines and chemokines, the authors saw an expected up-regulation of interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) and found that the most markedly enhanced chemokine was CXCL5—in both species. Not only was CXCL5 most elevated at the time of maximum pain, it was directly shown to be an important contributor to UVB-induced pain: Its injection alone into rat skin caused mechanical (but not thermal) hypersensitivity. CXCL5 attracted neutrophils and macrophages to the inflamed area. The final proof that CXCL5 is the key regulator for UVB-induced pain is that a neutralizing antibody to the chemokine protected against the pain and infiltration of immune cells. This fits with the known ability of CXCL5 to attract neutrophils (and as shown here macrophages) by regulating their chemotaxis. Pain is not always controlled in rodent models the same way that it is in humans. This has often prevented efficient translation of results in animal models to humans. Here, the authors guarded against this problem by showing a clear correlation of the UVB response in rat and in human skin, giving them confidence that their rat results would apply in humans. At least for inflammatory pain caused by the UVB rays of the sun, CXCL5 is an attractive target for therapeutic agents. 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.

A clinically relevant rodent model of the HIV antiretroviral drug stavudine induced painful peripheral neuropathy

Summary Rats treated with the HIV antiretroviral drug stavudine revealed alterations in complex ethologically relevant behaviours associated with pain and a distinct peripheral nerve neuropathology. Abstract HIV‐associated sensory neuropathy is the most frequent manifestation of HIV disease, afflicting 40–50% of patients whose HIV disease is otherwise controlled by antiretroviral therapy. It often presents with significant neuropathic pain and is consistently associated with previous exposure to nucleoside reverse transcriptase inhibitors including stavudine (d4T), which is widely used in resource‐limited settings. Here we investigated complex pain‐related behaviours associated with d4T treatment using ethologically relevant thigmotaxis and burrowing behaviours in adult rats. Detailed neuropathological response was also examined using neurochemistry, electron microscopy, and proteomics. After 2 intravenous injections of d4T (50 mg/kg, 4 days apart), rats developed hind paw mechanical hypersensitivity, which plateaued at 21 days after initial d4T injection, a time that these animals also had significant changes in thigmotaxis and burrowing behaviours when compared to the controls; reductions in hind paw intraepidermal nerve fibre density and CGRP/IB4 immunoreactivity in L5 spinal dorsal horn, suggesting injury to both the peripheral and central terminals of L5 dorsal root ganglion neurons; and increases in myelinated and unmyelinated axon diameters in the sural nerve, suggesting axonal swelling. However, no significant glial and inflammatory cell response to d4T treatment was observed. Sural nerve proteomics at 7 days after initial d4T injection revealed down‐regulated proteins associated with mitochondrial function, highlighting distal axons vulnerability to d4T neurotoxicity. In summary, we have reported complex behavioural changes and a distinctive neuropathology in a clinically relevant rat model of d4T‐induced sensory neuropathy that is suitable for further pathophysiological investigation and preclinical evaluation of novel analgesics.

A comparison of RNA-seq and exon arrays for whole genome transcription profiling of the L5 spinal nerve transection model of neuropathic pain in the rat

BackgroundThe past decade has seen an abundance of transcriptional profiling studies of preclinical models of persistent pain, predominantly employing microarray technology. In this study we directly compare exon microarrays to RNA-seq and investigate the ability of both platforms to detect differentially expressed genes following nerve injury using the L5 spinal nerve transection model of neuropathic pain. We also investigate the effects of increasing RNA-seq sequencing depth. Finally we take advantage of the “agnostic” approach of RNA-seq to discover areas of expression outside of annotated exons that show marked changes in expression following nerve injury.ResultsRNA-seq and microarrays largely agree in terms of the genes called as differentially expressed. However, RNA-seq is able to interrogate a much larger proportion of the genome. It can also detect a greater number of differentially expressed genes than microarrays, across a wider range of fold changes and is able to assign a larger range of expression values to the genes it measures. The number of differentially expressed genes detected increases with sequencing depth. RNA-seq also allows the discovery of a number of genes displaying unusual and interesting patterns of non-exonic expression following nerve injury, an effect that cannot be detected using microarrays.ConclusionWe recommend the use of RNA-seq for future high-throughput transcriptomic experiments in pain studies. RNA-seq allowed the identification of a larger number of putative candidate pain genes than microarrays and can also detect a wider range of expression values in a neuropathic pain model. In addition, RNA-seq can interrogate the whole genome regardless of prior annotations, being able to detect transcription from areas of the genome not currently annotated as exons. Some of these areas are differentially expressed following nerve injury, and may represent novel genes or isoforms. We also recommend the use of a high sequencing depth in order to detect differential expression for genes with low levels of expression.

Specific involvement of atypical PKCζ/PKMζ in spinal persistent nociceptive processing following peripheral inflammation in rat.

BackgroundCentral 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.ResultsUsing 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 Freunds 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.ConclusionsThese 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.

Altered potassium channel distribution and composition in myelinated axons suppresses hyperexcitability following injury

Neuropathic pain following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons, which begins to normalise over time. We investigated the composition and distribution of shaker-type-potassium channels (Kv1 channels) within the nodal complex of myelinated axons following injury. At the neuroma that forms after damage, expression of Kv1.1 and 1.2 (normally localised to the juxtaparanode) was markedly decreased. In contrast Kv1.4 and 1.6, which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in rats and humans. Within the dorsal root (a site remote from injury) we noted a redistribution of Kv1-channels towards the paranode. Blockade of Kv1 channels with α-DTX after injury reinstated hyperexcitability of A-fibre axons and enhanced mechanosensitivity. Changes in the molecular composition and distribution of axonal Kv1 channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. DOI: http://dx.doi.org/10.7554/eLife.12661.001