Kyungsoon Chung

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.

The effects of dorsal rhizotomy and spinal cord isolation on calcitonin gene-related peptide-labeled terminals in the rat lumbar dorsal horn☆

In the present study, the origin of calcitonin gene-related peptide (CGRP) to the dorsal horn in the rat lumbar spinal cord is investigated. CGRP immunoreactivity is examined following multiple unilateral and bilateral dorsal rhizotomies and isolated cord preparations (spinal cords are isolated by transecting the cord in two places and cutting all dorsal roots between the transections). Seven to 11 days after surgery, unilateral multiple dorsal rhizotomies result in a drastic decrease in CGRP-stained terminals on the operated side; following bilateral dorsal rhizotomies and isolated cord preparations, one or two CGRP varicosities remain in the dorsal horn in each section. The numbers of CGRP-immunostained varicosities observed in the latter two preparations are not significantly different, suggesting that few if any axons descending from the brain contribute to the CGRP terminal population in the spinal cord dorsal horn. Based on these data, we hypothesize that dorsal root ganglion cells are the only source of CGRP to the rat lumbar dorsal horn.

Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model

The extent of the sprouting of sympathetic postganglionic fibers in the dorsal root ganglion (DRG) and the peripheral nerves was examined in neuropathic rats at different postoperative times. After the L5 and L6 spinal nerves were ligated on one side, three different pain behavior tests (representing mechanical allodynia, cold allodynia, ongoing pain exacerbated by cold stress) were performed at various time intervals. The sympathetic postganglionic fibers were visualized by immunostaining with antibodies to tyrosine hydroxylase (TH). In the neuropathic rats, all three pain behaviors were fully developed within 3 days after the surgery, maintained up to 2 weeks, and then started to decline gradually afterward. At 20 weeks after neuropathic surgery, pain behaviors were reduced significantly compared to the peak response, but were still higher than the presurgery levels. Sympathectomy, performed 4 days after neuropathic surgery, almost completely abolished the signs of mechanical allodynia and ongoing pain behaviors, and it reduced the behaviors of cold allodynia to approximately half. The numerical density of sympathetic fibers in the DRG of an injured segment was significantly higher at 1, 4, and 20 weeks after neuropathic surgery as compared to the normal, suggesting that there is sprouting of sympathetic fibers in the DRG after peripheral nerve injury. Sprouting of sympathetic fibers in the DRG was extensive as early as 2 days after the spinal nerve ligation, and the sprouted fibers were almost completely eliminated after sympathectomy. The data suggest that sympathetic innervation of the DRG may play an important role in the development and maintenance of sympathetically maintained neuropathic pain.

The changes in expression of three subtypes of TTX sensitive sodium channels in sensory neurons after spinal nerve ligation.

Our previous studies showed that the ectopic discharges in injured sensory neurons and mechanical allodynia that developed after spinal nerve ligation were significantly reduced by application of a low concentration of tetrodotoxin (TTX) to the corresponding dorsal root ganglion (DRG) of the ligated spinal nerve. Based on these data, we hypothesized that expression of TTX-sensitive sodium channels is up-regulated in the injured sensory neurons and that such up-regulation plays an important role in the generation of ectopic discharges and thus pain behaviors in spinal nerve ligated neuropathic rats. To test this hypothesis, the present study examined the changes in three subtypes of TTX-sensitive sodium channels in the DRG after spinal nerve ligation. The changes in the total amount of mRNA for alpha-subunits of sodium channel brain type I (type I), brain type II (type II) and brain type III (type III) were determined by RNase protection assays (RPA). The population of DRG neurons expressing type III sodium channel protein was examined by an immunohistochemical method with antibodies to type III sodium channels. In the normal DRG, the level of mRNA for the type I sodium channel is high while that for type II and type III is very low. After spinal nerve ligation, the expression of type III mRNA was significantly increased at 16-h postoperatively (PO), doubled by 3 days PO and then was maintained at this high level until the end of the experiment (7 days PO). By contrast, the amount of mRNA for type I and type II sodium channels started to decrease at 1 day PO and were reduced to 25-50% of the normal control levels by 7 days after nerve ligation. Neurons showing positive immunostaining for type III sodium channels were rare ( approximately 3.2% of total population) in the normal DRG but increased after nerve ligation to 21% and 15% of the total neuronal population by 1 day and 7 days PO, respectively. Type III immunoreactivity was found preferentially in medium to large sized neurons. Thus the majority of neurons with cell bodies having diameters > or =40 microm became type III-positive after nerve ligation. The data indicate that the increased expression of type III sodium channels in axotomized sensory neurons may be the critical factor for the TTX sensitivity of ectopic discharges in injured sensory neurons and thus the generation of ectopic discharges and neuropathic pain behaviors in spinal nerve ligated rats.

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.

Abnormalities of sympathetic innervation in the area of an injured peripheral nerve in a rat model of neuropathic pain

Using the rat model that we have developed, a potential underlying mechanism for sympathetically maintained neuropathic pain (SMP) was explored. In rats showing neuropathic pain behaviors after a tight ligation of the L5 spinal nerve, putative sympathetic postganglionic fibers were examined in the injured spinal nerve and the dorsal root ganglion (DRG), using immunohistochemical staining with antibody against tyrosine hydroxylase (TH). In the neuropathic rats, there was an increase in the number of TH-immunolabeled fibers in the spinal nerve, and some DRG cells were surrounded by the labeled fibers. These abnormalities of sympathetic postganglionic innervation of the injured spinal nerve or the DRG may be a part of the mechanisms underlying the development of SMP.

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.

Low dose of tetrodotoxin reduces neuropathic pain behaviors in an animal model

We hypothesize that the accumulation of tetrodotoxin (TTX) sensitive sodium channels in injured dorsal root ganglion (DRG) neurons plays a critically important role in the generation of ectopic discharges and mechanical allodynia after peripheral nerve injury. Using the segmental spinal nerve (L5) ligation model of neuropathic pain, this hypothesis was tested by examining the effect of TTX on the mechanical sensitivity of the affected hind paw. Various concentrations of TTX were applied topically to the L5 DRG by using chronically implanted polyethylene tubing. The data showed that application of TTX at low doses (12.5-50 nM), which are far less than those needed for blocking action potential conduction, produced a significant elevation of mechanical threshold in the paw for foot withdrawals, a sign of reduced allodynic behaviors. The data suggest that TTX-sensitive subtypes of sodium channels play an important role in maintaining allodynic behaviors in an animal model of neuropathic pain.