Redox-basierte Mechanismen der Sensibilisierung bei neuropathischen Schmerzen

Bei Entzündung oder Verletzung peripherer Gewebe und Nerven kommt es zur Bildung reaktiver Sauerstoffspezies (ROS) im schmerzleitenden System. Welche ROS-generierenden Systeme hierbei beteiligt sind, ist jedoch nur ansatzweise verstanden. In der vorliegenden Arbeit konnte gezeigt werden, dass die ROS-produzierende NADPH Oxidase 4 (Nox4) einen wichtigen ROS-Generator im nozizeptiven System darstellt. Nox4 wird in unmyelinisierten nicht-peptidergen sowie in myelinisierten primär afferenten Neuronen exprimiert. In Modellen für akute und inflammatorische Schmerzen zeigten Nox4-/--Mäuse ein ähnliches Verhalten wie ihre wildtypischen Wurfgeschwister, jedoch war ihr Schmerzverhalten in Modellen für neuropathische Schmerzen reduziert. Eine Microarray-Analyse des lumbalen Rückenmarks nach peripherer Nervenverletzung zeigte eine Hochregulation der Expression Myelin-spezifischer Gene in Wildtyp-, nicht aber in Nox4-/- Mäusen. Darüber hinaus wurden in Wildtyp-Mäusen Myelin-spezifische Proteine im N. ischiadicus nach peripherer Nervenverletzung herab reguliert, während in Nox4-/--Mäusen keine Regulation dieser Proteine beobachtet wurde. Diese Ergebnisse deuten darauf hin, dass Nox4 eine essentielle Rolle bei Myelinisierungsprozessen spielt und so die Verarbeitung neuropathischer Schmerzsignale beeinflusst. Neben dem ROS-produzierenden System Nox4 wurde auch die Rolle des Peroxid-abbauenden Proteins Sestrin 2 (Sesn2) im nozizeptiven System untersucht. Nach peripherer Nervenverletzung wurde Sesn2-mRNA in den Spinalganglien und Sesn2-Protein im peripheren Nerv hochreguliert. Sesn2-/--Mäuse zeigten ein normales Verhalten in Modellen für akute und inflammatorische Schmerzen. Ihr Schmerzverhalten war jedoch im Formalin-Test und nach peripherer Nervenverletzung verstärkt. Diese Ergebnisse lassen vermuten, dass sowohl Nox4 als auch Sesn2 bei der Verarbeitung neuropathischer Schmerzsignale wichtige Funktionen einnehmen. Während Nox4 pronozizeptiv wirkt, weist Sesn2 antinozizeptive Effekte auf. Die Produktion reaktiver Sauerstoffspezies scheint daher ein wichtiger endogener Faktor der Sensibilisierung im Rahmen von neuropathischen Schmerzen zu sein.Damage or inflammation of tissues and peripheral nerves leads to the production of reactive oxygen species (ROS) in the nociceptive system. However, little is known about pain-relevant ROS generating systems. In the present work the ROS producing NADPH oxidase 4 (Nox4) was identified as an important ROS generator in the nociceptive system. Nox4 is expressed in a subset of nonpeptidergic nonmyelinated and myelinated primary afferent neurons. Nox4-/- mice demonstrated a normal nociceptive behavior in models of acute and inflammatory pain, however their nociceptive behavior was reduced in models of neuropathic pain. A microarray screen of the lumbal spinal cord after peripheral nerve injury revealed an induction of myelin specific genes in wildtype mice but not in Nox4-/- mice. Moreover, peripheral myelin specific proteins were downregulated in wildtype mice after peripheral nerve injury while there was no regulation of these proteins in Nox4-/- mice. These results indicate that Nox4 plays an essential role during myelination processes that may contribute to the processing of neuropathic pain. In addition to Nox4 the role of the peroxide degrading protein Sestrin 2 (Sesn2) in the nociceptive system was investigated. After peripheral nerve injury Sesn2 mRNA is induced in dorsal root ganglia and Sesn2 protein is upregulated in the peripheral nerve. Sesn2-/- mice demonstrated a normal behavior in models of acute and inflammatory pain. However, their neuropathic pain behavior was enhanced after peripheral nerve injury. These results suggest that Nox4 and Sesn2 essentially contribute to the processing of neuropathic pain. Nox4 acts in a pronociceptive manner, while Sesn2 demonstrates antinociceptive effects. Therefore the production of ROS seems to be an important endogenous factor that contributes to neuropathic pain processing.

Rab27a Contributes to the Processing of Inflammatory Pain in Mice

Tissue injury and inflammation may result in chronic pain, a severe debilitating disease that is associated with great impairment of quality of life. An increasing body of evidence indicates that members of the Rab family of small GTPases contribute to pain processing; however, their specific functions remain poorly understood. Here, we found using immunofluorescence staining and in situ hybridization that the small GTPase Rab27a is highly expressed in sensory neurons and in the superficial dorsal horn of the spinal cord of mice. Rab27a mutant mice, which carry a single-nucleotide missense mutation of Rab27a leading to the expression of a nonfunctional protein, show reduced mechanical hyperalgesia and spontaneous pain behavior in inflammatory pain models, while their responses to acute noxious mechanical and thermal stimuli is not affected. Our study uncovers a previously unrecognized function of Rab27a in the processing of persistent inflammatory pain in mice.publishersversionpublishe

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 Freund's 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

NADPH Oxidases in Pain Processing

Inflammation or injury to the somatosensory nervous system may result in chronic pain conditions, which affect millions of people and often cause major health problems. Emerging lines of evidence indicate that reactive oxygen species (ROS), such as superoxide anion or hydrogen peroxide, are produced in the nociceptive system during chronic inflammatory and neuropathic pain and act as specific signaling molecules in pain processing. Among potential ROS sources in the somatosensory system are NADPH oxidases, a group of electron-transporting transmembrane enzymes whose sole function seems to be the generation of ROS. Interestingly, the expression and relevant function of the Nox family members Nox1, Nox2, and Nox4 in various cells of the nociceptive system have been demonstrated. Studies using knockout mice or specific knockdown of these isoforms indicate that Nox1, Nox2, and Nox4 specifically contribute to distinct signaling pathways in chronic inflammatory and/or neuropathic pain states. As selective Nox inhibitors are currently being developed and investigated in various physiological and pathophysiological settings, targeting Nox1, Nox2, and/or Nox4 could be a novel strategy for the treatment of chronic pain. Here, we summarize the distinct roles of Nox1, Nox2, and Nox4 in inflammatory and neuropathic processing and discuss the effectiveness of currently available Nox inhibitors in the treatment of chronic pain conditions

Long-term treatment with low dose Vit C and Vit E does not inhibit CFA-induced inflammatory pain behavior.

<p>Mice were injected with 20 µl CFA into a hindpaw and drugs were i.p. administered at the indicated doses once daily for 12 days starting immediately after CFA injection. Paw withdrawal latency times upon mechanical stimulation are shown. Statistical analyses revealed no significant differences between groups. <i>n</i> = 8 per group.</p

CFA-induced inflammatory pain behavior is not affected by Vit C and Vit E treatment.

<p>Mice were injected with 20 µl CFA into a hindpaw. Drugs (15 mg Vit C, 7.5 mg Vit E, the combination of 15 mg Vit C and 7.5 mg Vit E, or saline) were i.p. administered 24 h (time point ‘0’) and 48 h (time point ‘24’) after CFA injection. Paw withdrawal latency times upon mechanical stimulation are expressed as difference to baseline (i.e. prior to the first drug injection). Statistical analyses revealed no significant differences between groups. <i>n</i> = 7–8 per group.</p

Vit C and Vit E pretreatment attenuates mechanical allodynia induced by intrathecal TBHP.

<p>Mice were i.p. pretreated with a combination of 30 mg Vit C and 15 mg Vit E or saline. One hour thereafter, the ROS donor TBHP (100 µg) was i.t. injected (time point ‘0’), and paw withdrawal latency times upon mechanical stimulation were measured for 105 min. <i>n</i> = 6 per group; *significantly different from saline group, <i>p</i><0.05.</p

Vit C and Vit E treatment attenuates phosphorylation of p38, but not of p42 or p44, in the spinal cord and DRGs.

<p>Mice were subjected to SNI surgery and received two i.p. injections of saline or the combination of Vit C (30 mg) and Vit E (15 mg) at days 14 and 15 after SNI. The protein expression of phospho-p38 (p-p38) and p-38 (A), and of phospho-p42 (p-p42), p42, phospho-p44 (p-p44) and p44 (B) in the spinal cord and DRGs was analyzed by western blotting of tissues obtained 3 h after the second drug injection. Calnexin was used as loading control. Representative western blots are shown on the left, densitometric analyses are shown on the right. <i>n</i> = 3 animals per group; *<i>p</i><0.05, **<i>p</i><0.01.</p

Antinociceptive effects of a Vit C and Vit E combination in the formalin test.

<p>Drugs (15 mg Vit C, 7.5 mg Vit E, the combination of 15 mg Vit C and 7.5 mg Vit E, or saline) were intraperitoneally administered 20 min prior to injection of formalin (15 µl, 5%) into a hindpaw. Sum of paw-licking time in phase 1 (1–10 min) and phase 2 (11–60 min). Note that the licking behavior in phase 1 is considerably reduced in mice treated with the Vit C+E combination. <i>n</i> = 6–8 per group; *significantly different from saline group, <i>p</i><0.05.</p