Marc R. Suter

p38 MAPK, microglial signaling, and neuropathic pain

Accumulating evidence over last several years indicates an important role of microglial cells in the pathogenesis of neuropathic pain. Signal transduction in microglia under chronic pain states has begun to be revealed. We will review the evidence that p38 MAPK is activated in spinal microglia after nerve injury and contributes importantly to neuropathic pain development and maintenance. We will discuss the upstream mechanisms causing p38 activation in spinal microglia after nerve injury. We will also discuss the downstream mechanisms by which p38 produces inflammatory mediators. Taken together, current data suggest that p38 plays a critical role in microglial signaling under neuropathic pain conditions and represents a valuable therapeutic target for neuropathic pain management.

JNK-Induced MCP-1 Production in Spinal Cord Astrocytes Contributes to Central Sensitization and Neuropathic Pain

Our previous study showed that activation of c-jun-N-terminal kinase (JNK) in spinal astrocytes plays an important role in neuropathic pain sensitization. We further investigated how JNK regulates neuropathic pain. In cultured astrocytes, tumor necrosis factor α (TNF-α) transiently activated JNK via TNF receptor-1. Cytokine array indicated that the chemokine CCL2/MCP-1 (monocyte chemoattractant protein-1) was strongly induced by the TNF-α/JNK pathway. MCP-1 upregulation by TNF-α was dose dependently inhibited by the JNK inhibitors SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) and D-JNKI-1. Spinal injection of TNF-α produced JNK-dependent pain hypersensitivity and MCP-1 upregulation in the spinal cord. Furthermore, spinal nerve ligation (SNL) induced persistent neuropathic pain and MCP-1 upregulation in the spinal cord, and both were suppressed by D-JNKI-1. Remarkably, MCP-1 was primarily induced in spinal cord astrocytes after SNL. Spinal administration of MCP-1 neutralizing antibody attenuated neuropathic pain. Conversely, spinal application of MCP-1 induced heat hyperalgesia and phosphorylation of extracellular signal-regulated kinase in superficial spinal cord dorsal horn neurons, indicative of central sensitization (hyperactivity of dorsal horn neurons). Patch-clamp recordings in lamina II neurons of isolated spinal cord slices showed that MCP-1 not only enhanced spontaneous EPSCs but also potentiated NMDA- and AMPA-induced currents. Finally, the MCP-1 receptor CCR2 was expressed in neurons and some non-neuronal cells in the spinal cord. Together, we have revealed a previously unknown mechanism of MCP-1 induction and action. MCP-1 induction in astrocytes after JNK activation contributes to central sensitization and neuropathic pain facilitation by enhancing excitatory synaptic transmission. Inhibition of the JNK/MCP-1 pathway may provide a new therapy for neuropathic pain management.

Blocking Caspase Activity Prevents Transsynaptic Neuronal Apoptosis and the Loss of Inhibition in Lamina II of the Dorsal Horn after Peripheral Nerve Injury

We show that transsynaptic apoptosis is induced in the superficial dorsal horn (laminas I-III) of the spinal cord by three distinct partial peripheral nerve lesions: spared nerve injury, chronic constriction, and spinal nerve ligation. Ongoing activity in primary afferents of the injured nerve and glutamatergic transmission cause a caspase-dependent degeneration of dorsal horn neurons that is slow in onset and persists for several weeks. Four weeks after spared nerve injury, the cumulative loss of dorsal horn neurons, determined by stereological analysis, is >20%. GABAergic inhibitory interneurons are among the neurons lost, and a marked decrease in inhibitory postsynaptic currents of lamina II neurons coincides with the induction of apoptosis. Blocking apoptosis with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) prevents the loss of GABAergic interneurons and the reduction of inhibitory currents. Partial peripheral nerve injury results in pain-like behavioral changes characterized by hypersensitivity to tactile or cold stimuli. Treatment with zVAD, which has no intrinsic analgesic properties, attenuates this neuropathic pain-like syndrome. Preventing nerve injury-induced apoptosis of dorsal horn neurons by blocking caspase activity maintains inhibitory transmission in lamina II and reduces pain hypersensitivity.

Do glial cells control pain

Management of chronic pain is a real challenge, and current treatments that focus on blocking neurotransmission in the pain pathway have resulted in limited success. Activation of glial cells has been widely implicated in neuroinflammation in the CNS, leading to neurodegeneration in conditions such as Alzheimers disease and multiple sclerosis. The inflammatory mediators released by activated glial cells, such as tumor necrosis factor-a and interleukin-1b not only cause neurodegeneration in these disease conditions, but also cause abnormal pain by acting on spinal cord dorsal horn neurons in injury conditions. Pain can also be potentiated by growth factors such as brain-derived growth factor and basic fibroblast growth factor, which are produced by glia to protect neurons. Thus, glial cells can powerfully control pain when they are activated to produce various pain mediators. We review accumulating evidence that supports an important role for microglial cells in the spinal cord for pain control under injury conditions (e.g. nerve injury). We also discuss possible signaling mechanisms, in particular mitogen-activated protein kinase pathways that are crucial for glial-mediated control of pain.Investigating signaling mechanisms in microglia might lead to more effective management of devastating chronic pain.

Glial Cells and Chronic Pain

Over the past few years, the control of pain exerted by glial cells has emerged as a promising target against pathological pain. Indeed, changes in glial phenotypes have been reported throughout the entire nociceptive pathway, from peripheral nerves to higher integrative brain regions, and pharmacological inhibition of such glial reactions reduces the manifestation of pain in animal models. This complex interplay between glia and neurons relies on various mechanisms depending both on glial cell types considered (astrocytes, microglia, satellite cells, or Schwann cells), the anatomical location of the regulatory process (peripheral nerve, spinal cord, or brain), and the nature of the chronic pain paradigm. Intracellularly, recent advances have pointed to the activation of specific cascades, such as mitogen-associated protein kinases (MAPKs) in the underlying processes behind glial activation. In addition, given the large number of functions accomplished by glial cells, various mechanisms might sensitize nociceptive neurons including a release of pronociceptive cytokines and neurotrophins or changes in neurotransmitter-scavenging capacity. The authors review the conceptual advances made in the recent years about the implication of central and peripheral glia in animal models of chronic pain and discuss the possibility to translate it into human therapies in the future.

Nerve conduction blockade in the sciatic nerve prevents but does not reverse the activation of p38 mitogen-activated protein kinase in spinal microglia in the rat spared nerve injury model.

Background: Current evidence indicates that p38 mitogen-activated protein kinase activation in spinal microglia contributes to the development of neuropathic pain. However, how nerve injury activates p38 in spinal microglia is incompletely unknown. Nerve injury–induced ectopic spontaneous activity is essential for the generation of neuropathic pain. The authors examined whether peripheral neural activity is necessary for p38 activation in spinal microglia. Methods: To examine whether spinal microglia activation depends on peripheral activity in the rat spared nerve injury (SNI) model, the authors blocked conduction in the sciatic nerve before or 2 days after SNI. The block was produced by applying bupivacaine-loaded microspheres above the nerve injury site. The p38 activation was examined by p38 phosphorylation using a phosphorylated p38 antibody, and neuropathic pain-related behavior was evaluated before and after intrathecal infusion of a p38 inhibitor. Results: Three days after SNI, there was a marked p38 activation in the medial two thirds of the dorsal horn, where the injured tibial and peroneal nerves terminated and where isolectin B4 staining was lost. Phosphorylated p38 was only colocalized with the microglial surface marker OX-42, indicating a microglial localization of phosphorylated p38 in the SNI model. Bupivacaine microspheres produced persistent block (loss of sensory and motor function) of the sciatic nerve for the whole period of the study (3 days). This blockade prevented but did not reverse p38 activation in spinal microglia. Intrathecal infusion of the p38 inhibitor FR167653 prevented and reversed mechanical allodynia on post-SNI day 3. Conclusions: After nerve injury, activity in the peripheral nerve is required for the induction but not the maintenance of p38 activation in spinal microglia.

Activation of p38 mitogen-activated protein kinase in spinal microglia contributes to incision-induced mechanical allodynia.

Background:Recent studies have implicated the activation of stress-activated mitogen-activated protein kinase (MAPK) p38 in spinal microglial cells for development of neuropathic and inflammatory pain. The aim of the present study was to investigate whether phosphorylation of p38 (p-p38) also mediates mechanical allodynia and thermal hyperalgesia induced by plantar incision. Methods:After rats received a plantar incision surgery, mechanical allodynia and thermal hyperalgesia were determined by von Frey filaments and radiant heat, respectively, and the number of p-p38 immunoreactive cells in the dorsal horn was quantified to determine p38 activation at different time points after incision. The p38 inhibitor FR167653 was administered intrathecally 30 min before hind paw plantar incision to determine the role of p38 in postoperative pain. Results:A significant increase in number of p-p38 immunoreactive cells was observed in the ipsilateral L4-5 spinal dorsal horn from 1 h to 3 days after the incision. p-p38 was found predominantly in microglia. However, microglial activation (assessed by OX-42 upregulation) was not evident until 3 days after plantar incision. Intrathecal pretreatment of FR167653 attenuated incision-induced mechanical allodynia from 1 h to day 2 and significantly reduced activation of p38 in the dorsal horn 1 day after plantar incision. However, FR167653 only inhibited heat hyperalgesia at an early time point. Conclusions:Plantar incision-induced mechanical allodynia can be prevented by the p38 inhibitor. Our results suggest that p38 activation in spinal microglia play a role in incision-induced mechanical allodynia in rats. Therefore, p38 inhibition may be useful in treating postsurgical pain.

Large A-fiber activity is required for microglial proliferation and p38 MAPK activation in the spinal cord: different effects of resiniferatoxin and bupivacaine on spinal microglial changes after spared nerve injury

BackgroundAfter peripheral nerve injury, spontaneous ectopic activity arising from the peripheral axons plays an important role in inducing central sensitization and neuropathic pain. Recent evidence indicates that activation of spinal cord microglia also contributes to the development of neuropathic pain. In particular, activation of p38 mitogen-activated protein kinase (MAPK) in spinal microglia is required for the development of mechanical allodynia. However, activity-dependent activation of microglia after nerve injury has not been fully addressed. To determine whether spontaneous activity from C- or A-fibers is required for microglial activation, we used resiniferatoxin (RTX) to block the conduction of transient receptor potential vanilloid subtype 1 (TRPV1) positive fibers (mostly C- and Aδ-fibers) and bupivacaine microspheres to block all fibers of the sciatic nerve in rats before spared nerve injury (SNI), and observed spinal microglial changes 2 days later.ResultsSNI induced robust mechanical allodynia and p38 activation in spinal microglia. SNI also induced marked cell proliferation in the spinal cord, and all the proliferating cells (BrdU+) were microglia (Iba1+). Bupivacaine induced a complete sensory and motor blockade and also significantly inhibited p38 activation and microglial proliferation in the spinal cord. In contrast, and although it produced an efficient nociceptive block, RTX failed to inhibit p38 activation and microglial proliferation in the spinal cord.Conclusion(1) Blocking peripheral input in TRPV1-positive fibers (presumably C-fibers) is not enough to prevent nerve injury-induced spinal microglial activation. (2) Peripheral input from large myelinated fibers is important for microglial activation. (3) Microglial activation is associated with mechanical allodynia.

Development of neuropathic pain in the rat spared nerve injury model is not prevented by a Peripheral nerve block

Background The mechanisms responsible for initiation of persistent neuropathic pain after peripheral nerve injury are unclear. One hypothesis is that injury discharge and early ectopic discharges in injured nerves produce activity-dependent irreversible changes in the central nervous system. The aim of this study was to determine whether blockade of peripheral discharge by blocking nerve conduction before and 1 week after nerve injury could prevent the development and persistence of neuropathic pain–like behavior in the spared nerve injury model. Methods Bupivacaine-loaded biodegradable microspheres embedded in fibrin glue were placed in a silicone tube around the sciatic nerve to produce a conduction block. After sensory–motor testing of block efficacy, a spared nerve injury procedure was performed. Development of neuropathic pain behavior was assessed for 4 weeks by withdrawal responses to stimulation (i.e., von Frey filaments, acetone, pinprick, radiant heat) in bupivacaine microspheres–treated animals (n = 12) and in controls (n = 11). Results Bupivacaine microspheres treatment produced conduction blockade with a complete lack of sensory responsiveness in the sural territory for 6 to 10 days. Once the block wore off, the degree of hypersensitivity to stimuli was similar in both groups. Conclusions Peripheral long-term nerve blockade has no detectable effect on the development of allodynia or hyperalgesia in the spared nerve injury model. It is unlikely that injury discharge at the time of nerve damage or the early onset of ectopic discharges arising from the injury site contributes significantly to the persistence of stimulus-evoked neuropathic pain in this model.

Molecular evidence of late preconditioning after sevoflurane inhalation in healthy volunteers.

BACKGROUND:Late preconditioning by volatile anesthetics evolves in response to transcriptional changes. We hypothesized that sevoflurane inhalation would modify the transcriptome in human blood and modulate the expression of adhesion molecules in white blood cells consistent with the occurrence of a late preconditioning phase. METHODS:Five healthy male subjects inhaled sevoflurane at an end-tidal concentration of 0.5%–1.0% for 60 min. Venous blood samples were collected at baseline, after 15 and 60 min of inhalation, and 6, 24, 48, and 72 h thereafter and immediately processed for flow cytometry and mRNA extraction and hybridization to Affymetrix U133 Plus 2.0 microarrays. Data were analyzed using Significance Analysis of Microarray and Gene Set Enrichment Analysis and confirmed by real-time reverse transcription polymerase chain reaction. L-selectin (CD62L) and &bgr;2-integrin (CD11b) expression was determined on granulocytes and monocytes using flow cytometry. RESULTS:Sevoflurane inhalation rapidly and markedly altered gene expression in white blood cells. Key transcripts potentially involved in late preconditioning or organ protection including paraoxonase, 12-lipoxygenase, heat shock protein 40, chemokine ligand 5, and phosphodiesterase 5A were regulated in response to sevoflurane. Sevoflurane further decreased transcripts involved in peroxisome proliferator-activated receptor &ggr; coactivator-1&agr; (PGC-1&agr;) signaling and fatty acid oxidation. Reduced L-selectin (CD62L) expression on granulocytes accompanied with increased resistance to inflammatory activation was present at 24 to 48 h after sevoflurane exposure. CONCLUSIONS:Sevoflurane at subanesthetic concentrations modifies blood transcriptome and decreases the expression of the proinflammatory L-selectin (CD62L), consistent with a “second window of protection” in humans.