Wiebke Kallenborn-Gerhardt

NADPH Oxidase-4 Maintains Neuropathic Pain after Peripheral Nerve Injury

Reactive oxygen species (ROS) contribute to sensitization of pain pathways during neuropathic pain, but little is known about the primary sources of ROS production and how ROS mediate pain sensitization. Here, we show that the NADPH oxidase isoform Nox4, a major ROS source in somatic cells, is expressed in a subset of nonpeptidergic nociceptors and myelinated dorsal root ganglia neurons. Mice lacking Nox4 demonstrated a substantially reduced late-phase neuropathic pain behavior after peripheral nerve injury. The loss of Nox4 markedly attenuated injury-induced ROS production and dysmyelination processes of peripheral nerves. Moreover, persisting neuropathic pain behavior was inhibited after tamoxifen-induced deletion of Nox4 in adult transgenic mice. Our results suggest that Nox4 essentially contributes to nociceptive processing in neuropathic pain states. Accordingly, inhibition of Nox4 may provide a novel therapeutic modality for the treatment of neuropathic pain.

NOXious signaling in pain processing

Chronic pain affects millions of people and often causes major health problems. Accumulating evidence indicates that the production of reactive oxygen species (ROS), such as superoxide anion or hydrogen peroxide, is increased in the nociceptive system during chronic inflammatory and neuropathic pain, and that ROS can act as specific signaling molecules in pain processing. Reduction of ROS levels by administration of scavengers or antioxidant compounds attenuated the nociceptive behavior in various animal models of chronic pain. However, the sources of increased ROS production during chronic pain and the role of ROS in pain processing are poorly understood. Current work revealed pain-relevant functions of the Nox family of NADPH oxidases, a group of electron-transporting transmembrane enzymes whose sole function seems to be the generation of ROS. In particular, significant expression of the Nox family members Nox1, Nox2, and Nox4 in various cells of the nociceptive system has been discovered. Studies using knockout mice suggest that these Nox enzymes specifically contribute to distinct signaling pathways in chronic inflammatory and/or neuropathic pain states. Accordingly, targeting Nox1, Nox2, and Nox4 could be a novel strategy for the treatment of chronic pain. Currently selective inhibitors of Nox enzymes are being developed. Here, we introduce the distinct roles of Nox enzymes in pain processing, we summarize recent findings in the understanding of ROS-dependent signaling pathways in the nociceptive system, and we discuss potential analgesic properties of currently available Nox inhibitors.

Additive antinociceptive effects of a combination of vitamin C and vitamin E after peripheral nerve injury.

Accumulating evidence indicates that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic pain. We show that systemic administration of a combination of vitamins C and E inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freunds Adjuvant. In contrast, vitamin C or vitamin E given alone failed to affect the nociceptive behavior in all tested models. The attenuated neuropathic pain behavior induced by the vitamin C and E combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states.

CNGA3: A Target of Spinal Nitric Oxide/cGMP Signaling and Modulator of Inflammatory Pain Hypersensitivity

A large body of evidence indicates that nitric oxide (NO) and cGMP contribute to central sensitization of pain pathways during inflammatory pain. Here, we investigated the distribution of cyclic nucleotide-gated (CNG) channels in the spinal cord, and identified the CNG channel subunit CNGA3 as a putative cGMP target in nociceptive processing. In situ hybridization revealed that CNGA3 is localized to inhibitory neurons of the dorsal horn of the spinal cord, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. CNGA3 expression is upregulated in the superficial dorsal horn of the mouse spinal cord and in dorsal root ganglia following hindpaw inflammation evoked by zymosan. Mice lacking CNGA3 (CNGA3−/− mice) exhibited an increased nociceptive behavior in models of inflammatory pain, whereas their behavior in models of acute or neuropathic pain was normal. Moreover, CNGA3−/− mice developed an exaggerated pain hypersensitivity induced by intrathecal administration of cGMP analogs or NO donors. Our results provide evidence that CNGA3 contributes in an inhibitory manner to the central sensitization of pain pathways during inflammatory pain as a target of NO/cGMP signaling.

Slack Channels Expressed in Sensory Neurons Control Neuropathic Pain in Mice

Slack (Slo2.2) is a sodium-activated potassium channel that regulates neuronal firing activities and patterns. Previous studies identified Slack in sensory neurons, but its contribution to acute and chronic pain in vivo remains elusive. Here we generated global and sensory neuron-specific Slack mutant mice and analyzed their behavior in various animal models of pain. Global ablation of Slack led to increased hypersensitivity in models of neuropathic pain, whereas the behavior in models of inflammatory and acute nociceptive pain was normal. Neuropathic pain behaviors were also exaggerated after ablation of Slack selectively in sensory neurons. Notably, the Slack opener loxapine ameliorated persisting neuropathic pain behaviors. In conclusion, Slack selectively controls the sensory input in neuropathic pain states, suggesting that modulating its activity might represent a novel strategy for management of neuropathic pain.

Nox2-dependent signaling between macrophages and sensory neurons contributes to neuropathic pain hypersensitivity.

&NA; Nox2 produces reactive oxygen species in dorsal root ganglia macrophages after peripheral nerve injury, thereby contributing to TNF&agr;‐dependent neuropathic pain signaling. &NA; Emerging lines of evidence indicate that production of reactive oxygen species (ROS) at distinct sites of the nociceptive system contributes to the processing of neuropathic pain. However, the mechanisms underlying ROS production during neuropathic pain processing are not fully understood. We here detected the ROS‐generating nicotinamide adenine dinucleotide phosphate oxidase isoform Nox2 in macrophages of dorsal root ganglia (DRG) in mice. In response to peripheral nerve injury, Nox2‐positive macrophages were recruited to DRG, and ROS production was increased in a Nox2‐dependent manner. Nox2‐deficient mice displayed reduced neuropathic pain behavior after peripheral nerve injury, whereas their immediate responses to noxious stimuli were normal. Moreover, injury‐induced upregulation of tumor necrosis factor &agr; was absent, and activating transcription factor 3 induction was reduced in DRG of Nox2‐deficient mice, suggesting an attenuated macrophage‐neuron signaling. These data suggest that Nox2‐dependent ROS production in macrophages recruited to DRG contributes to neuropathic pain hypersensitivity, underlining the observation that Nox‐derived ROS exert specific functions during the processing of pain.

Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury

AIMS Emerging lines of evidence indicate that oxidants such as hydrogen peroxide exert specific signaling functions during the processing of chronic pain. However, the mechanisms by which oxidants regulate pain processing in vivo remain poorly understood. Here, we investigated whether cyclic guanosine monophosphate (cGMP)-dependent protein kinase Iα (cGKIα), which can be activated by oxidants independently of cGMP, serves as a primary redox target during pain processing. RESULTS After peripheral nerve injury, oxidant-induced cGKIα activation is increased in dorsal root ganglia of mice. Knock-in (KI) mice in which cGKIα cannot transduce oxidant signals demonstrated reduced neuropathic pain behaviors after peripheral nerve injury, and reduced pain behaviors after intrathecal delivery of oxidants. In contrast, acute nociceptive, inflammatory, and cGMP-induced pain behaviors were not impaired in these mice. INNOVATION Studying cGKIα KI mice, we provide the first evidence that oxidants activate cGKIα in sensory neurons after peripheral nerve injury in vivo. CONCLUSION Our results suggest that oxidant-induced activation of cGKIα specifically contributes to neuropathic pain processing, and that prevention of cGKIα redox activation could be a potential novel strategy to manage neuropathic pain.

Lack of effect of a P2Y6 receptor antagonist on neuropathic pain behavior in mice

Accumulating evidence indicates that various subtypes of purinergic receptors (P2X and P2Y receptor families) play an essential role in the development and the maintenance of neuropathic pain. However, there is only limited data available about the role of P2Y6 receptors in pain processing. Here we detected P2Y6 receptor immunoreactivity in primary afferent neurons of mice and observed an upregulation in response to peripheral nerve injury. However, systemic and intrathecal administration of the P2Y6 receptor antagonist MRS2578 failed to affect the injury-induced neuropathic pain behavior. Our results suggest that P2Y6 receptors, in contrast to other purinergic receptor subtypes, are not critically involved in nerve injury-induced neuropathic pain processing in mice.

Phosphodiesterase 2A Localized in the Spinal Cord Contributes to Inflammatory Pain Processing

Background:Phosphodiesterase 2A (PDE2A) is an evolutionarily conserved enzyme that catalyzes the degradation of the cyclic nucleotides, cyclic adenosine monophosphate, and/or cyclic guanosine monophosphate. Recent studies reported the expression of PDE2A in the dorsal horn of the spinal cord, pointing to a potential contribution to the processing of pain. However, the functions of PDE2A in spinal pain processing in vivo remained elusive. Methods:Immunohistochemistry, laser microdissection, and quantitative real-time reverse transcription polymerase chain reaction experiments were performed to characterize the localization and regulation of PDE2A protein and messenger RNA in the mouse spinal cord. Effects of the selective PDE2A inhibitor, BAY 60–7550 (Cayman Chemical, Ann Arbor, MI), in animal models of inflammatory pain (n = 6 to 10), neuropathic pain (n = 5 to 6), and after intrathecal injection of cyclic nucleotides (n = 6 to 8) were examined. Also, cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in spinal cord tissues were measured by liquid chromatography tandem mass spectrometry. Results:The authors here demonstrate that PDE2A is distinctly expressed in neurons of the superficial dorsal horn of the spinal cord, and that its spinal expression is upregulated in response to hind paw inflammation. Administration of the selective PDE2A inhibitor, BAY 60–7550, increased the nociceptive behavior of mice in animal models of inflammatory pain. Moreover, BAY 60–7550 increased the pain hypersensitivity induced by intrathecal delivery of cyclic adenosine monophosphate, but not of cyclic guanosine monophosphate, and it increased the cyclic adenosine monophosphate levels in spinal cord tissues. Conclusion:Our findings indicate that PDE2A contributes to the processing of inflammatory pain in the spinal cord.

The H2S-producing enzyme CSE is dispensable for the processing of inflammatory and neuropathic pain

Accumulating lines of evidence indicate that hydrogen sulfide (H2S) contributes to the processing of chronic pain. However, the sources of H2S production in the nociceptive system are poorly understood. Here we investigated the expression of the H2S releasing enzyme cystathionine γ-lyase (CSE) in the nociceptive system and characterized its role in chronic pain signaling using CSE deficient mice. We show that paw inflammation and peripheral nerve injury led to upregulation of CSE expression in dorsal root ganglia. However, conditional knockout mice lacking CSE in sensory neurons as well as global CSE knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to WT littermates. Thus, our results suggest that CSE is not critically involved in chronic pain signaling in mice and that sources different from CSE mediate the pain relevant effects of H2S.