Hee Kee Kim

Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain

&NA; Reactive oxygen species (ROS) are free radicals produced in biological systems that are involved in various degenerative brain diseases. The present study tests the hypothesis that ROS also play an important role in neuropathic pain. In the rat spinal nerve ligation (SNL) model of neuropathic pain, mechanical allodynia develops fully 3 days after nerve ligation and persists for many weeks. Systemic injection of a ROS scavenger, phenyl‐N‐tert‐butylnitrone (PBN), relieves SNL‐induced mechanical allodynia in a dose‐dependent manner. Repeated injections cause no development of tolerance or no loss of potency. Preemptive treatment with PBN is also effective in preventing full development of neuropathic pain behavior. Systemic injection was mimicked by intrathecal injection with a little less efficacy, while intracerebroventricular administration produced a much smaller effect. These data suggest that PBN exerts its anti‐allodynic action mainly by spinal mechanisms. Systemic treatment with other spin‐trap reagents, 5,5‐dimethylpyrroline‐N‐oxide and nitrosobenzene, showed similar analgesic effects, suggesting that ROS are critically involved in the development and maintenance of neuropathic pain. Thus this study suggests that systemic administration of non‐toxic doses of free radical scavengers could be useful for treatment of neuropathic pain.

Reactive oxygen species (ROS) are involved in enhancement of NMDA-receptor phosphorylation in animal models of pain

Abstract Recent studies indicate that reactive oxygen species (ROS) play an important role in neuropathic pain, predominantly through spinal mechanisms. Since the data suggest that ROS are involved in central sensitization, the present study examines the levels of activated N‐methyl‐d‐aspartate (NMDA) receptors in the dorsal horn before and after removal of ROS with a ROS scavenger, phenyl‐N‐t‐butyl nitrone (PBN), in animal models of pain. Tight ligation of the L5 spinal nerve was used for the neuropathic pain model and intradermal injection of capsaicin was used for the inflammatory pain model. Foot withdrawal thresholds to von Frey stimuli to the paw were measured as pain indicators. The number of neurons showing immunoreactivity to phosphorylated NMDA‐receptor subunit 1 (pNR1) and the total amount of pNR1 proteins in the spinal cord were determined using immunohistochemical and Western blotting techniques, respectively. Hyperalgesia and increased pNR1 expression were observed in both neuropathic and capsaicin‐treated rats. A systemic injection of PBN (100 mg/kg, i.p.) dramatically reduced hyperalgesia and blocked the enhancement of spinal pNR1 in both pain models within 1 h after PBN treatment. The data suggest that ROS are involved in NMDA‐receptor activation, an essential step in central sensitization, and thus contribute to neuropathic and capsaicin‐induced pain.

The role of reactive oxygen species in capsaicin-induced mechanical hyperalgesia and in the activities of dorsal horn neurons.

Abstract Previous findings that reactive oxygen species (ROS) are involved in neuropathic pain, mainly through spinal mechanisms, suggest that ROS may be involved in central sensitization. To investigate the possible role of ROS in central sensitization, we examined in rats the effects of ROS scavengers on capsaicin‐induced secondary hyperalgesia, which is known to be mediated by central sensitization. We used two different ROS scavengers: phenyl N‐tert‐butylnitrone (PBN) and 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (TEMPOL). Intradermal capsaicin injection (20 μg in 20 μl olive oil) into the hind paw produced primary and secondary hyperalgesia. A systemic administration of PBN (100 mg/kg, i.p.) or TEMPOL (200 mg/kg, i.p.) alleviated capsaicin‐induced secondary, but not primary, hyperalgesia. Intrathecal injection of PBN (1 mg inof vertinary Surgery/anesthesiology, College of vetrinary Medic 50 μl saline) greatly reduced hyperalgesia, whereas intracerebroventricular or intradermal injection of PBN produced only a minor analgesic effect, suggesting that PBN takes effect mainly through the spinal cord. Electrophysiological recordings from wide dynamic range (WDR) neurons in the dorsal horn showed that intradermal capsaicin enhanced the evoked responses to peripheral stimuli; systemic PBN or TEMPOL restored the responses to normal levels. Removal of ROS thus restored the responsiveness of spinal WDR neurons to normal levels, suggesting that ROS is involved in central sensitization, at least in part by sensitizing WDR neurons.

Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats

&NA; NR1 is an essential component of functional NMDA receptors and can be activated by phosphorylation. It is suggested that phosphorylation of NR1 (pNR1) contributes to central sensitization after intradermal capsaicin injection. The present study investigates whether increases of spinal pNR1 are correlated to central sensitization and thus pain behaviors in neuropathic pain. Neuropathic rats were produced by L5 spinal nerve ligation, mechanical thresholds of the paw were measured, and then the L4/5 spinal cords and the nucleus gracilis (NG) were removed and immunostained for pNR1. The results showed that the number of pNR1‐immunoreactive neurons was significantly increased in the ipsilateral cord, at 3, 7, and 28 days after nerve ligation and these increases coincide with mechanical allodynia. The increase of pNR1‐immunoreactive neurons in the NG was observed only at 28 days after the nerve ligation. Western blot analyses confirmed the significant increase of pNR1 protein in spinal dorsal horn after nerve ligation. A protein kinase A inhibitor, H89, moderately reversed mechanical allodynia in 7 day neuropathic rats. Many pNR1‐immunoreactive neurons were identified as projection neurons by retrograde tracer. The data suggest that PKA mediated NMDA receptor phosphorylation plays an important role in spinal nerve ligation induced neuropathic pain.

Reactive oxygen species contribute to neuropathic pain by reducing spinal GABA release

&NA; Although both a loss of spinal inhibitory neurotransmission and the involvement of oxidative stress have been regarded as important mechanisms in the pathogenesis of pain, the relationship between these 2 mechanisms has not been studied. To determine whether reactive oxygen species (ROS) involvement in pain mechanisms is related to the diminished inhibitory transmission in the substantia gelatinosa (SG) of the spinal dorsal horn, behavioral studies and whole‐cell recordings were performed in FVB/NJ mice. Neuropathic pain was induced by a tight ligation of the L5 spinal nerve (SNL). Pain behaviors in the affected foot were assessed by behavioral testing for mechanical hyperalgesia. Pain behaviors developed by 3 days and lasted more than 8 weeks. Both systemic and intrathecal administration of an ROS scavenger, phenyl‐N‐tert‐butylnitrone (PBN), temporarily reversed mechanical hyperalgesia up to 2 hours, 1 week after SNL. In nonligated mice, an intrathecal injection of an ROS donor, tert‐butyl hydroperoxide (t‐BOOH), dose‐dependently induced mechanical hyperalgesia for 1.5 hours. In whole‐cell voltage clamp recordings of SG neurons, perfusion with t‐BOOH significantly decreased the frequency of mIPSCs, and this effect was reversed by PBN. Furthermore, t‐BOOH decreased the frequency of GABAA receptor‐mediated mIPSCs without altering their amplitudes but did not affect glycine receptor‐mediated mIPSCs. In SNL mice, mIPSC frequency in SG neurons was significantly reduced as compared with that of normal mice, which was restored by PBN. The antihyperalgesic effect of PBN on mechanical hyperalgesia was attenuated by intrathecal bicuculline, a GABAA receptor blocker. Our results indicate that the increased ROS in spinal cord may induce pain by reducing GABA inhibitory influence on SG neurons that are involved in pain transmission. An increase in ROS in spinal cord may induce pain by reducing GABA inhibitory influence on substantia gelatinosa neurons that are involved in pain transmission.

Analgesic effect of vitamin E is mediated by reducing central sensitization in neuropathic pain.

Abstract Recent studies suggest that reactive oxygen species (ROS) are critically involved in neuropathic pain. Although vitamin E is a well‐known antioxidant, its efficacy on chronic pain is not known. This study investigated the efficacy and mechanisms of vitamin E analgesia in a rat model of neuropathic pain produced by spinal nerve ligation. The effects of vitamin E were investigated using behavioral testing, electrophysiological recording of dorsal horn neurons, and determinations of phosphorylated NMDA receptor subunit 1 (pNR1) levels in the spinal dorsal horn. Results showed that a systemic single injection of a high dose or repetitive daily injections of low doses of vitamin E significantly reduced neuropathic pain behaviors. Vitamin E was also effective in producing analgesia by intrathecal injection, suggesting the importance of spinal mechanisms. In spinal dorsal horn neurons, vitamin E reduced evoked responses to mechanical stimuli as well as the sizes of their receptive fields. In addition, levels of pNR1 in neuropathic rats were also reduced by vitamin E injection. These data suggest that vitamin E produces analgesia in neuropathic rats that is, at least in part, mediated by reducing central sensitization which, in turn, is induced by peripheral nerve injury.

Segmental Spinal Nerve Ligation Model of Neuropathic Pain

Since its introduction in 1992, the spinal nerve ligation (SNL) model of neuropathic pain has been widely used for various investigative works on neuropathic pain mechanisms as well as in screening tests for the development of new analgesic drugs. This model was developed by tightly ligating one (L5) or two (L5 and L6) segmental spinal nerves in the rat. The operation results in long-lasting behavioral signs of mechanical allodynia, heat hyperalgesia, cold allodynia, and ongoing pain. In the process of widespread usage, however, many different variations of the SNL model have been produced, either intentionally or unintentionally, by different investigators. Although the factors that cause these variations themselves are interesting and important topics to be studied, the pain mechanisms involved in these variations are likely different from the original model. Therefore, this chapter describes, in detail, the method for producing the spinal nerve ligation model that will minimally induce potential factors that may contribute to these variations. It is hoped that this description will help many investigators to produce a consistent animal model with uniform pathophysiological mechanisms.

Injury Discharges Regulate Calcium Channel Alpha-2-Delta-1 Subunit Upregulation in the Dorsal Horn that Contributes to Initiation of Neuropathic Pain

Abstract Previous studies have shown that peripheral nerve injury in rats induces increased expression of the voltage gated calcium channel (VGCC) alpha‐2‐delta‐1 subunit (Cavα2δ1) in spinal dorsal horn and sensory neurons in dorsal root ganglia (DRG) that correlates to established neuropathic pain states. To determine if injury discharges trigger Cavα2δ1 induction that contributes to neuropathic pain initiation, we examined allodynia onset and Cavα2δ1 levels in DRG and spinal dorsal horn of spinal nerve ligated rats after blocking injury induced neural activity with a local brief application of lidocaine on spinal nerves before the ligation. The lidocaine pretreatment blocked ligation‐induced discharges in a dose‐dependent manner. Similar pretreatment with the effective concentration of lidocaine diminished injury‐induced increases of the Cavα2δ1 in DRG and abolished that in spinal dorsal horn specifically, and resulted in a delayed onset of tactile allodynia post‐injury. Both dorsal horn Cavα2δ1 upregulation and tactile allodynia in the lidocaine pretreated rats returned to levels similar to that in saline pretreated controls 2 weeks post the ligation injury. In addition, preemptive intrathecal Cavα2δ1 antisense treatments blocked concurrently injury‐induced allodynia onset and Cavα2δ1 upregulation in dorsal spinal cord. These findings indicate that injury induced discharges regulate Cavα2δ1 expression in the spinal dorsal horn that is critical for neuropathic allodynia initiation. Thus, preemptive blockade of injury‐induced neural activity or Cavα2δ1 upregulation may be a beneficial option in neuropathic pain management.

Phenyl N-tert-butylnitrone, a free radical scavenger, reduces mechanical allodynia in chemotherapy-induced neuropathic pain in rats

Background:Paclitaxel is a widely used chemotherapeutic drug for breast and ovarian cancer. Unfortunately, it induces neuropathic pain, which is a dose-limiting side effect. Free radicals have been implicated in many neurodegenerative diseases. The current study tests the hypothesis that a free radical scavenger plays an important role in reducing chemotherapy-induced neuropathic pain. Methods:Neuropathic pain was induced by intraperitoneal injection of paclitaxel (2 mg/kg) on four alternate days (days 0, 2, 4, and 6) in male Spraue-Dawley rats. Phenyl N-tert-butylnitrone (PBN), a free radical scavenger, was administered intraperitoneally as a single dose or multiple doses before or after injury. Mechanical allodynia was measured by using von Frey filaments. Results:The administration of paclitaxel induced mechanical allodynia, which began to manifest on days 7–10, peaked within 2 weeks, and plateaued for at least 2 months after the first paclitaxel injection. A single injection or multiple intraperitoneal injections of PBN ameliorated paclitaxel-induced pain behaviors in a dose-dependent manner. Further, multiple administrations of PBN starting on day 7 through day 15 after the first injection of paclitaxel completely prevented the development of mechanical allodynia. However, an intraperitoneal administration of PBN for 8 days starting with the first paclitaxel injection did not prevent the development of pain behavior. Conclusions:This study clearly shows that PBN alleviated mechanical allodynia induced by paclitaxel in rats. Furthermore, our data show that PBN given on days 7 through 15 after the first paclitaxel injection prevented the development of chemotherapy-induced neuropathic pain. This clearly has a clinical implication.

Electroacupuncture suppresses capsaicin-induced secondary hyperalgesia through an endogenous spinal opioid mechanism.

ABSTRACT Central sensitization, caused either by tissue inflammation or peripheral nerve injury, plays an important role in persistent pain. An animal model of capsaicin‐induced pain has well‐defined peripheral and central sensitization components, thus is useful for studying the analgesic effect on two separate components. The focus of this study is to examine the analgesic effects of electroacupuncture (EA) on capsaicin‐induced secondary hyperalgesia, which represents central sensitization. Capsaicin (0.1%, 20 μl) was injected into the plantar side of the left hind paw, and foot withdrawal thresholds in response to von Frey stimuli (mechanical sensitivity) were determined for both primary and secondary hyperalgesia in rats. EA (2 Hz, 3 mA) was applied to various pairs of acupoints, GB30–GB34, BL40–BL60, GV2–GV6, LI3–LI6 and SI3–TE8, for 30 min under isoflurane anesthesia and then the effect of EA on mechanical sensitivity of paw was determined. EA applied to the ipsilateral SI3–TE8, but to none of the other acupoints, significantly reduced capsaicin‐induced secondary hyperalgesia but not primary hyperalgesia. EA analgesic effect was inhibited by a systemic non‐specific opioid receptor (OR) antagonist or an intrathecal μ‐ or &dgr;‐OR antagonist. EA analgesic effect was not affected by an intrathecal κ‐OR antagonist or systemic adrenergic receptor antagonist. This study demonstrates that EA produces a stimulation point‐specific analgesic effect on capsaicin‐induced secondary hyperalgesia (central sensitization), mediated by activating endogenous spinal μ‐ and &dgr;‐opioid receptors.