Search

This study shows that the direction of synaptic plastic changes in the spinal cord is cell-type specific in response to nociceptive input., The underlying mechanism of chronic pain is believed to be changes in excitability in spinal dorsal horn (DH) neurons that respond abnormally to peripheral input. Increased excitability in pain transmission neurons, and depression of inhibitory neurons, are widely recognized in the spinal cord of animal models of chronic pain. The possible occurrence of 2 parallel but opposing forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) was tested in 2 types of identified DH neurons using whole-cell patch-clamp recordings in mouse spinal cord slices. The test stimulus was applied to the sensory fibers to evoke excitatory postsynaptic currents in identified spinothalamic tract neurons (STTn) and GABAergic neurons (GABAn). Afferent conditioning stimulation (ACS) applied to primary afferent fibers with various stimulation parameters induced LTP in STTn but LTD in GABAn, regardless of stimulation parameters. These opposite responses were further confirmed by simultaneous dual patch-clamp recordings of STTn and GABAn from a single spinal cord slice. Both the LTP in STTn and the LTD in GABAn were blocked by an NMDA receptor antagonist, AP5, or an intracellular Ca2+ chelator, BAPTA. Both the pattern and magnitude of intracellular Ca2+ after ACS were almost identical between STTn and GABAn based on live-cell calcium imaging. The results suggest that the intense sensory input induces an NMDA receptor-dependent intracellular Ca2+ increase in both STTn and GABAn, but produces opposing synaptic plasticity. This study shows that there is cell type–specific synaptic plasticity in the spinal DH.

The increase of cytosolic free Ca2+([Ca2+]c) due to NMDA receptor activation is a key step for spinal cord synaptic plasticity by altering cellular signal transduction pathways. We focus on this plasticity as a cause of persistent pain. To provide a mechanism for these classic findings, we report that [Ca2+]cdoes not trigger synaptic plasticity directly but must first enter into mitochondria. Interfering with mitochondrial Ca2+uptake during a [Ca2+]cincrease blocks induction of behavioral hyperalgesia and accompanying downstream cell signaling, with reduction of spinal long-term potentiation (LTP). Furthermore, reducing the accompanying mitochondrial superoxide levels lessens hyperalgesia and LTP induction. These results indicate that [Ca2+]crequires downstream mitochondrial Ca2+uptake with consequent production of reactive oxygen species (ROS) for synaptic plasticity underlying chronic pain. These results suggest modifying mitochondrial Ca2+uptake and thus ROS as a type of chronic pain therapy that should also have broader biologic significance.

Acute ankle injuries are common problems and often lead to persistent pain. To investigate the underlying mechanism of ankle sprain pain, the response properties of spinal dorsal horn neurons were examined after ankle sprain. Acute ankle sprain was induced manually by overextending the ankle of a rat hindlimb in a direction of plantarflexion and inversion. The weight-bearing ratio (WBR) of the affected foot was used as an indicator of pain. Single unit activities of dorsal horn neurons in response to plantarflexion and inversion of the foot or ankle compression were recorded from the medial part of the deep dorsal horn, laminae IV-VI, in normal and ankle-sprained rats. One day after ankle sprain, rats showed significantly reduced WBRs on the affected foot, and this reduction was partially restored by systemic morphine. The majority of deep dorsal horn neurons responded to a single ankle stimulus modality. After ankle sprain, the mean evoked response rates were significantly increased, and afterdischarges were developed in recorded dorsal horn neurons. The ankle sprain-induced enhanced evoked responses were significantly reduced by morphine, which was reversed by naltrexone. The data indicate that movement-specific dorsal horn neuron responses were enhanced after ankle sprain in a morphine-dependent manner, thus suggesting that hyperactivity of dorsal horn neurons is an underlying mechanism of pain after ankle sprain.

Acupuncture, an alternative medical therapy with a long history, is appealing because it can activate endogenous analgesic mechanisms by minimally invasive means. The mechanisms of acupuncture, however, are not well understood yet. The following sentence was removed from our original manuscript. One of the major problems impeding understanding of the acupuncture mechanism is lack of experimental models that mimic various forms of persistent pain that respond to acupuncture in humans.In this review, we summarize and discuss previous and recent findings regarding electroacupuncture-induced analgesia in an ankle sprain pain model and the potential underlying mechanisms of acupuncture.A novel model of ankle sprain pain is introduced recently and the mechanism of electroacupuncture-induced analgesia in this model has been explored. The following sentence was removed from our original manuscript. This model provides a reproducible and quantifiable index of persistent pain at the ankle joint in rats. Acupuncture at a remote site produces long-lasting and powerful analgesia. The consistent analgesic effect of acupuncture in this model has allowed us to pursue the underlying neural mechanisms.These studies provide insight into the mechanisms of acupuncture analgesia in one particular form of persistent pain, and hopefully will allow us to expand our knowledge to other painful conditions.

Two reactive oxygen species (ROS), nitric oxide (NO•) and superoxide (•O2−), contribute to persistent pain. Using three different animal models where ROS mediate pain, this study examined whether NO• and •O2− converge to peroxynitrite (ONOO−) or whether each has an independent signaling pathway to produce hyperalgesia. The hyperalgesia following spinal nerve ligation was attenuated by removing superoxide by TEMPOL or inhibiting NO• production by L-NAME, but not by removing peroxynitrite with FeTMPyP. Nitric oxide-induced hyperalgesia was not affected by removing superoxide but was reduced by a guanyl cyclase inhibitor. Superoxide-induced hyperalgesia was not affected by inhibiting NO• production but was suppressed by a PKC inhibitor. The data suggest that nitric oxide andsuperoxide operate independently to generate pain.

Reactive oxygen species (ROS) scavengers have been shown to relieve persistent pain; however, the mechanism is not clearly understood. Superoxide produced from mitochondrial oxidative phosphorylation is considered the major source of ROS in neurons during excitation where mitochondrial superoxide levels are normally controlled by superoxide dismutase (SOD-2). The present study hypothesizes that capsaicin-induced secondary hyperalgesia is a consequence of superoxide build-up in spinal dorsal horn neurons and SOD-2 is a major determinant. To test this hypothesis, the spinal levels of SOD-2 activity, inactivated SOD-2 proteins, and mitochondrial superoxide were measured and correlated to the levels of capsaicin-induced secondary hyperalgesia in mice with and without SOD-2 manipulations. The data suggest that superoxide accumulation is a culprit in the abnormal sensory processing in the spinal cord in capsaicin-induced secondary hyperalgesia. Our studies also support the notion that SOD-2 nitration is a critical mechanism that maintains elevated superoxide levels in the spinal cord after capsaicin treatment. Finally, our findings suggest a therapeutic potential for the manipulation of spinal SOD-2 activity in pain conditions.

Scavengers of reactive oxygen species (ROS) have been shown to produce a strong antinociceptive effect on persistent pain, and mitochondria are suggested to be the main source of ROS in the spinal dorsal horn. To explore whether excessive generation of mitochondrial superoxide alone can induce pain, the effect of mitochondrial electron transport complex inhibitors on the development of mechanical hyperalgesia was examined in mice. Intrathecal injection of an electron transport complex inhibitor, antimycin A or rotenone, in normal mice resulted in a slowly developing but long-lasting and dose-dependent mechanical hyperalgesia. The levels of mechanical hyperalgesia after antimycin A, a complex III inhibitor, were higher than that with rotenone, a complex I inhibitor. A large increase of mitochondrial superoxide in the spinal dorsal horn and a strong antinociceptive effect of ROS scavengers, phenyl-N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were observed in antimycin A-treated mice. The study indicates that the enhanced production of spinal mitochondrial superoxide alone without nerve injury can produce mechanical hyperalgesia.

To examine a possible involvement of reactive oxygen species (ROS) in visceral pain, the levels of ROS in the colon and the effect of a ROS scavenger phenyl N-t-butylnitrone (PBN) on pain were examined in zymosan-induced colitis rats. Zymosan was instilled into the colon of adult rats. The electromyograms (EMGs) of abdominal muscle contractions in response to colorectal distension (CRD) were recorded as an indicator of visceral pain. After zymosan treatment, the rats showed enhanced EMG and elevated levels of H2O2 in the colon. PBN treatment (intraperitoneal, intrathecal or intracolonic) significantly reduced the enhanced EMGs induced by zymosan. The results suggest that elevated ROS in the spinal cord and the colon are involved in visceral pain.

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 microg in 20 microl olive oil) into the hind paw produced primary and secondary hyperalgesia. A systemic administration of PBN (100mg/kg, i.p.) or TEMPOL (200mg/kg, i.p.) alleviated capsaicin-induced secondary, but not primary, hyperalgesia. Intrathecal injection of PBN (1mg inof veterinary Surgery/anesthesiology, College of veterinary Medic 50 microl 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.

Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among ≈1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

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.

Increased purinergic sensitivity of injured sensory neurons suggests the possible involvement of purinoceptors for the generation of pain after nerve injury. To identify the purinoceptors that are involved, the changes in mRNA levels of 6 subtype purinoceptors were examined in the dorsal root ganglia (DRG) of the normal rat and after spinal nerve ligation, using RNase protection assay (RPA). In addition, the P2X 2 containing neurons were examined in the L5 DRG, using an immunohistochemical method. The relative amounts of mRNAs for the six purinoceptor subtypes were in the order of P2X 3 ⪢P2X 4 >P2X 6 >P2X 5 ≈P2X 2 >P2X 1 in the normal lumbar DRG. After nerve injury, the mRNA of P2X 5 was increased, those of P2X 3 and P2X 6 were decreased, and those of P2X 2 and P2X 4 were unchanged. Immunohistochemical studies, however, showed 23% of the total DRG neurons are P2X 2 positive in the normal L5 DRG, but that increased to 73% after nerve ligation. These data suggest that not only transcriptional but also posttranscriptional changes of multiple purinoceptors might be involved in the enhancement of purinergic sensitivity in injured sensory neurons.

Purinoceptors are present in the cell bodies as well as in both peripheral and central terminals of many sensory neurons, where they may play a role in sensory transmission, including pain. After peripheral nerve injury at the spinal nerve level, some axotomized afferent neurons develop ongoing discharges (ectopic discharges) that originate in the dorsal root ganglion (DRG). In the present study, we attempted to determine whether or not purinergic sensitivity develops in injured sensory neurons which display ectopic discharges, as well as in silent units. The L(4) and L(5) spinal nerves were ligated in Sprague-Dawley rats. Four to 21 days after the surgery, the DRGs with attached dorsal roots and spinal nerves were removed and ectopic discharges were recorded from teased dorsal root fascicles using an in vitro recording set-up. The results showed that 75.6 and 65.1% of the chronically axotomized DRG neurons displaying ectopic discharges enhanced their activity after application of adenosine 5'-triphosphate (ATP, 1 mM) or alpha,beta-methylene ATP (mATP, 100 microM), respectively. In addition, application of these purinoceptor agonists evoked activity in 7 of 28 axotomized DRG neurons, which did not show ongoing discharges. In contrast, only 1 of 34 DRG neurons acutely isolated from normal rats (no previous spinal nerve ligation) responded to either mATP or ATP. In most of the tested units, mATP-induced enhancement of ectopic discharges was blocked by non-specific P2X receptor antagonists, PPADS or suramin. The data from the present study suggest that purinergic sensitivity develops in DRG neurons after chronic axotomy and that this purinergic sensitivity is likely to be mediated by P2X purinoceptors. This acquired purinergic sensitivity may play an important functional role in the enhancement of ectopic discharges and exacerbation of pain upon sympathetic activation in the neuropathic pain state.

To evaluate the sympathetic dependency of pain behaviors in an animal model of neuropathic pain, the effect of surgical sympathectomy on the mechanical sensitivity of the hindpaw was examined in rats with L5 spinal nerve ligation. Mechanical sensitivity was determined by measuring foot withdrawal thresholds to mechanical stimulation with von Frey filaments applied to the base of the third or fourth toe. Tight ligation of the segmental L5 spinal nerve led to the development of mechanical hypersensitivity in the hindpaw. The effects of 2 different procedures of surgical lumbar sympathectomy on mechanical hypersensitivity were compared, limited (resection of sympathetic chain/ganglia L2 to L4 segments) and extensive (resection of L2 to L6 segments) sympathectomies. Mechanical hypersensitivity produced by L5 spinal nerve ligation was partially but significantly reduced by both sympathectomy procedures. In a separate group of rats, the L5 spinal nerve was ligated while irritating the neighboring L4 spinal nerve. This procedure produced a lesser degree of mechanical hypersensitivity, and subsequent sympathectomy had no effect on these animals. These data suggest that sympathectomy is effective in this model only when the animals show severe mechanical hypersensitivity.

In spinal nerve ligated Lewis strain neuropathic rats, pain behaviors and the rate of ectopic discharges of injured sensory neurons were significantly reduced by systemic injection of phentolamine. A pharmacological study indicated that this adrenergic dependency was mediated by alpha(1)-adrenoceptors (alpha(1)-AR). The development of adrenergic sensitivity in injured sensory neurons might have resulted from changes in adrenoceptor expression as a consequence of changed expression of adrenoceptor genes. This possibility was examined by determining the changes in the mRNA expression of 3 subtypes of alpha(1)-ARs, alpha(1a)-, alpha(1b)-, and alpha(1d)-ARs, in the dorsal root ganglia (DRG) after spinal nerve ligation. The L4 and L5 spinal nerves were tightly ligated in Lewis rats. One week later, the L4 and L5 DRG were collected and RNase protection assay (RPA) and in situ hybridization were performed. In the DRG of unoperated rats, a moderate amount of alpha(1a)-AR mRNA was present while the amount of either alpha(1b)-AR or alpha(1d)-AR mRNA was small. After spinal nerve ligation, there was a significant increase in the amount of alpha(1b)-AR mRNA in the nerve ligated DRG as measured by RPA. The amount of alpha(1a)-AR mRNA was decreased to 20% of the normal level while that of alpha(1d)-AR mRNA did not change. The in situ hybridization study showed that the number of alpha(1b)-AR mRNA positive neurons increased in spinal nerve ligated DRG, confirming the results of RPA study. These data suggest that the up-regulated expression of alpha(1b)-AR mRNA in axotomized DRG neurons may play an important role in the development of adrenergic sensitivity in injured sensory neurons and thus contribute to the sympathetically maintained pain in spinal nerve ligated neuropathic Lewis rats.

In an attempt to identify important ion channels contributing to the generation of ectopic discharges, the present study examined the effects of ion channel blockers on ectopic discharges of injured sensory neurons after spinal nerve ligation. The main focus of the study was to examine the effect of the sodium channel blocker, tetrodotoxin (TTX), in order to identify important subtype(s) (i.e. TTX-sensitive and TTX-resistant) of sodium channels that are involved in ectopic discharge generation. In addition, the effects of potassium and calcium channel blockers were also tested for comparison with the results of previous studies. The dorsal root ganglion (DRG) of the injured segment was removed along with the dorsal root (DR) and the spinal nerve 7-14 days after spinal nerve ligation in the rat. The tissue was placed in an in-vitro recording chamber consisting of multiple compartments that were independently perfused with 35 degrees C artificial cerebrospinal fluid (ACSF). Single unit recordings were made from teased DR fibers. Once a spontaneously active unit was found and characterized, ACSF containing a channel blocker was perfused to the DRG, the site where almost all ectopic discharges originate after spinal nerve ligation. All the recorded spontaneously active units were found to be Abeta and Adelta fibers (no C fibers were detected). Perfusion of the DRG with a sodium channel blocker (lidocaine) at a dose much less than that required to block conduction of action potentials, significantly inhibited ectopic discharges in all recorded fibers. In addition, ectopic discharges were inhibited by TTX perfused to the DRG at a dose much lower (average of 22.1 nM) than that required to block TTX-resistant subtypes of sodium channels. The data suggest that TTX-sensitive sodium channels are likely to be involved in the generation of ectopic discharges. The present study also confirmed the results of previous studies on the additional potential roles of potassium and calcium channels, thus suggesting that multiple ion channels are likely to be involved in the generation of ectopic discharges.

Calcitonin gene-related peptide (CGRP) is a vasodilatory peptide, and it is primarily synthesized in dorsal root ganglia (DRG). Plasma CGRP levels increase during pregnancy and with steroid hormones, and nerve growth factor (NGF) stimulates calcitonin/CGRP promoter and CGRP synthesis in DRG. We previously showed that CGRP levels in DRG were stimulated with steroid hormone treatments in vivo but not in vitro. Thus, the stimulation of CGRP by these hormones may be indirect through the upregulation of NGF effects. We hypothesized that the female sex steroid hormones upregulate NGF receptors, trkA and p75(NTR), in DRG. We examined the effects of 17 beta-estradiol (E(2)) and progesterone (P(4)) on NGF receptors in DRG obtained from ovariectomized (ovx) rats. Groups of 4 ovx rats were injected s.c. with 5 microg E(2), 4 mg P(4), or 5 microg E(2) + 4 mg P(4) in 0.2 ml sesame oil or injected with oil only and were killed at 6, 24, and 48 h. In addition, ovx rats were also injected s.c. with varying doses (0.2, 1.0, 5.0, 25 microg) of E(2) (0.5, 1.5, 4, 10 mg) P(4), and (5 microg) E(2) + (0.5, 1.5, 4.0, 10 mg) P(4) in 0.2 ml sesame oil, or vehicle, and killed at 6 (for E(2)) or 24 (for P(4) and E(2) + P(4)) h. Furthermore, groups of ovx rats were also killed at 12 and 24 h; 3 and 7 days; 2, 4, and 6 wk after ovariectomy. The DRGs were collected from all groups and then processed for Western immunoblotting to examine both trkA and p75(NTR) levels. Estradiol increased trkA at 6 h but not p75(NTR). Progesterone caused upregulation of trkA and p75(NTR) at 6 and 24 h. 17 beta-Estradiol + P(4) increased trkA at 6 and 24 h and p75(NTR) at all time points examined. One microgram of E(2) increased trkA but did not affect p75(NTR) levels. Progesterone at 4 and 10 mg upregulated trkA but only 10 mg P(4) increased p75(NTR). Five micrograms of E(2) coinjected with P(4) at 1.5 and 4 mg increased trkA, while p75(NTR) receptor was upregulated when coinjected with P(4) at 1.5 to 10 mg. The ovariectomy caused a decrease in trkA receptors compared to proestrus rats, and these decreases were significant by 6 wk, but surprisingly p75(NTR) increased at 2 wk after ovariectomy. 17 beta-Estradiol increased trkA but not p75(NTR) receptors in DRG, whereas P(4) caused increases in both trkA and p75(NTR) in DRG. In addition, the combination of these steroid hormones had more effect on both receptors than either hormone alone. Thus, we concluded that high levels of female steroid hormones such as those due to pregnancy or hormonal replacement therapy could increase NGF receptor expression in DRG that carry more NGF to elevate the CGRP synthesis in these groups. We suggested that the regulation of NGF receptors by ovarian steroids may underlie steroidal regulation of other factors such as CGRP.

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.

This study compared and contrasted the manifestation of neuropathic pain behaviors in several strains of rats. These included ACI, Brown-Norway, Fischer 344, Lewis, Long-Evans, Sprague-Dawley, and Wistar-Furth, all obtained from Harlan Sprague-Dawley Inc. Comparison was also made between two substrains of Sprague-Dawley rats: one from Harlan and the other from Sasco. Neuropathic injury was produced by tightly ligating the left L5 and L6 spinal nerves with the animals under halothane anesthesia. Tests were conducted for 2 weeks to examine behavioral signs representing mechanical allodynia, cold allodynia, and spontaneous pain. There was no difference between strains in any of the tested behaviors before surgery. After neuropathic injury, rats in most groups developed high levels of behavioral signs of various components of neuropathic pain; however, some strains of rats showed weak behavioral signs of neuropathic pain. When a comparison was made between two substrains of Sprague-Dawley rats from two different sources, the ones from Sasco showed weaker behavioral signs than those from Harlan. When comparisons were made between different strains of rats from the same source (Harlan), Brown-Norway and Long-Evans rats showed the smallest magnitude of neuropathic pain behaviors. The data indicate that different strains and substrains of rats display different degrees of pain behaviors, suggesting that strains and substrains are important variables in the development of neuropathic pain after peripheral nerve injury.

Neurotrophins have specificity toward distinct subpopulations of dorsal root ganglion (DRG) neurons with different neurotrophin receptors. It has been suggested that neurotrophins also play important roles in mature DRG neurons after injury. In the present study, we examined the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) mRNAs in the DRG after a peripheral nerve injury. The data showed that following a spinal nerve ligation, the level of NGF mRNA increased 4 times over the normal level and was maintained at a high level for a period of 3 weeks. The induction of BDNF mRNA was brief (lasting less than 3 days) and lesser in quantity ( approximately 1. 7 times increase) compared to NGF expression. The expression of NT-3 mRNA was not detected either in normal or nerve injured rats. Results suggest that different neurotrophins play different functional roles in the DRG after spinal nerve injury.

Rat spinal neurons expressing lectin RL-29 are visualized immunohistochemically. RL-29 immunoreactive (RL-29 IR) neurons are found in the lateral parts of laminae V-VII, designated as the intermediolateral cell column (IML) in the thoracic cord, the sacral parasympathetic nucleus (SPN) in the lumbosacral cord, and the dorsomedial nucleus (DMN) of the ventral horn. The majority of RL-29 IR neurons in the SPN are also labeled by a retrograde tracer DAPI applied to the cut L6-S1 ventral roots. These data indicate that the majority of RL-29 IR neurons in the SPN are autonomic preganglionic neurons, thus suggesting that RL-29 can be a useful tool in marking this subpopulation of neurons. In addition, the presence of previously described RL-29 IR primary afferent fibers and terminals in the dorsal parts of the cord are confirmed.

One feature of neuropathic pain is a reduced spinal gamma-aminobutyric acid (GABA)-ergic inhibitory function. However, the mechanisms behind this attenuation remain to be elucidated. This study investigated the involvement of reactive oxygen species in the spinal GABA neuron loss and reduced GABA neuron excitability in spinal nerve ligation (SNL) model of neuropathic pain in mice. The importance of spinal GABAergic inhibition in neuropathic pain was tested by examining the effects of intrathecally administered GABA receptor agonists and antagonists in SNL and naive mice, respectively. The effects of SNL and antioxidant treatment on GABA neuron loss and functional changes were examined in transgenic GAD67-enhanced green fluorescent protein positive (EGFP+) mice. GABA receptor agonists transiently reversed mechanical hypersensitivity of the hind paw in SNL mice. On the other hand, GABA receptor antagonists made naive mice mechanically hypersensitive. Stereological analysis showed that the numbers of enhanced green fluorescent protein positive (EGFP+) GABA neurons were significantly decreased in the lateral superficial laminae (I-II) on the ipsilateral L5 spinal cord after SNL. Repeated antioxidant treatments significantly reduced the pain behaviors and prevented the reduction in EGFP+ GABA neurons. The response rate of the tonic firing GABA neurons recorded from SNL mice increased with antioxidant treatment, whereas no change was seen in those recorded from naive mice, which suggested that oxidative stress impaired some spinal GABA neuron activity in the neuropathic pain condition. Together the data suggest that neuropathic pain, at least partially, is attributed to oxidative stress, which induces both a GABA neuron loss and dysfunction of surviving GABA neurons.

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.

Background: Central sensitization-associated synaptic plasticity in the spinal cord dorsal horn (SCDH) critically contributes to the development of chronic pain, but understanding of the underlying molecular pathways is still incomplete. Emerging evidence suggests that Wnt signaling plays a crucial role in regulation of synaptic plasticity. Little is known about the potential function of the Wnt signaling cascades in chronic pain development. Results: Fluorescent immunostaining results indicate that β-catenin, an essential protein in the canonical Wnt signaling pathway, is expressed in the superficial layers of the mouse SCDH with enrichment at synapses in lamina II. In addition, Wnt3a, a prototypic Wnt ligand that activates the canonical pathway, is also enriched in the superficial layers. Immunoblotting analysis indicates that both Wnt3a a β-catenin are up-regulated in the SCDH of various mouse pain models created by hind-paw injection of capsaicin, intrathecal (i.t.) injection of HIV-gp120 protein or spinal nerve ligation (SNL). Furthermore, Wnt5a, a prototypic Wnt ligand for non-canonical pathways, and its receptor Ror2 are also up-regulated in the SCDH of these models. Conclusion: Our results suggest that Wnt signaling pathways are regulated by nociceptive input. The activation of Wnt signaling may regulate the expression of spinal central sensitization during the development of acute and chronic pain.

Sensitization of dorsal horn neurons (DHNs) in the spinal cord is dependent on pain-related synaptic plasticity and causes persistent pain. The DHN sensitization is mediated by a signal transduction pathway initiated by the activation of N-methyl-d-aspartate receptors (NMDA-Rs). Recent studies have shown that elevated levels of reactive oxygen species (ROS) and phosphorylation-dependent trafficking of GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) are a part of the signaling pathway for DHN sensitization. However, the relationship between ROS and AMPA-R phosphorylation and trafficking is not known. Thus, this study investigated the effects of ROS scavengers on the phosphorylation and cell-surface localization of GluA1 and GluA2. Intrathecal NMDA- and intradermal capsaicin-induced hyperalgesic mice were used for this study since both pain models share the NMDA-R activation-dependent DHN sensitization in the spinal cord. Our behavioral, biochemical, and immunohistochemical analyses demonstrated that: 1) NMDA-R activation in vivo increased the phosphorylation of AMPA-Rs at GluA1 (S818, S831, and S845) and GluA2 (S880) subunits; 2) NMDA-R activation in vivo increased cell-surface localization of GluA1 but decreased that of GluA2; and 3) reduction of ROS levels by ROS scavengers PBN (N-tert-butyl-α-phenylnitrone) or TEMPOL (4-hydroxy-2, 2, 6, 6-tetramethylpiperidin-1-oxyl) reversed these changes in AMPA-Rs, as well as pain-related behavior. Given that AMPA-R trafficking to the cell surface and synapse is regulated by NMDA-R activation-dependent phosphorylation of GluA1 and GluA2, our study suggests that the ROS-dependent changes in the phosphorylation and cell-surface localization of AMPA-Rs are necessary for DHN sensitization and thus, pain-related behavior. We further suggest that ROS reduction will ameliorate these molecular changes and pain.

The present study demonstrates that afferent fibers in the rat ventral root sprout after transection of the neonatal sciatic nerve. The key data are that the number of unmyelinated fibers increased dramatically in the L5 ventral roots and about half of these unmyelinated fibers were labeled with antibody to growth associated phosphoprotein (GAP-43), suggesting that they are regenerating axons. Furthermore, these fibers survived in the distal stump of the acutely cut ventral root, suggesting that they are afferent fibers. The results provide direct evidence for sprouting of afferent fibers in the ventral root induced by a neonatal peripheral nerve lesion.

Little information is available concerning the pelvic visceral afferent system, in view of its terminal location in the spinal cord and its associated transmitter substances in the rat. By utilizing an immunostaining method to examine the transneuronal neurotransmitter depletion resulting from peripheral sensory nerve injury, the spinal projections of primary afferent fibers containing calcitonin gene-related peptide (CGRP) and originating from pelvic viscera were studied in the lumbosacral spinal cord of the rat. After unilateral or bilateral pelvic nerve section, CGRP immunoreactivity in the lumbosacral spinal cord was decreased greatly in the sacral parasympathetic nucleus (SPN), the dorsolateral fasiculus, the medial border of the dorsal horn, the dorsal gray commissure (DGC), and the intermediate gray connecting the SPN and DGC. Fine structural analysis showed that the CGRP-immunoreactive terminals made synaptic contact with dendrites and, rarely, with somata. Although there was some incidence of a synaptic contact between a CGRP-IR terminal and a vesicle-containing profile, definite evidence of axo-axonal synapse has not been confirmed. These data indicate that CGRP-containing pelvic visceral primary afferent fibers project to autonomic areas of the lumbosacral spinal cord by way of the pelvic nerve and make synaptic contact with dendrites and somata.

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 two 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 three days and lasted over eight weeks. Both systemic and intrathecal administration of an ROS scavenger, phenyl-N-tert-butylnitrone (PBN), temporarily reversed mechanical hyperalgesia up to two hours one week after SNL. In non-ligated 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 to that of normal mice, which was restored by PBN. The anti-hyperalgesic 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.

The present study provides further evidence in support of the hypothesis that there is a fine primary afferent system in the dorsal funiculi by determining the effects of capsaicin (8-methyl-N-vanillyl-6-noneamide) on unmyelinated fibers in the cervical fasciculus gracilis of the rat. The neurolytic effect of this procedure was demonstrated by showing an 89% decrease in the number of unmyelinated fibers in the S2 dorsal roots of the experimental animals. Consequently, we feel that unmyelinated primary afferent fibers are largely removed from these animals. Neonatal administration of capsaicin (50 mg/kg) caused a 54% decrease in the number of unmyelinated fibers in the C3 fasciculus gracilis but no significant change in myelinated fiber numbers. The data provide further evidence for the existence of a significant primary afferent unmyelinated fiber system in the dorsal funiculus and suggest a role for the dorsal funiculi in the transmission of noxious information.

The present study is a quantitative analysis of the unmyelinated fiber population in the fasciculus gracilis of the second cervical segment of cat and monkey. We find that unmyelinated fibers represent 13.7% of the total fiber population in this pathway in the cat and 18.9% in the monkey (Macaca fascicularis). The existence of such large numbers of these axons suggests that there may be a sizeable ascending fine primary afferent pathway in the fasciculus gracilis in cat and monkey whose destination is presumably the dorsal column nuclei. These findings are of interest in regard to classic ideas that the afferent fibers in the dorsal columns are large myelinated fibers that convey fine discriminative information to the dorsal column nuclei.

Recent studies suggest that reactive oxygen species (ROS) are functional messenger molecules in central sensitization, an underlying mechanism of persistent pain. Because spinal cord long-term potentiation (LTP) is the electrophysiological basis of central sensitization, this study investigates the effects of the increased or decreased spinal ROS levels on spinal cord LTP. Spinal cord LTP is induced by either brief, high-frequency stimulation (HFS) of a dorsal root at C-fiber intensity or superfusion of a ROS donor, tert-butyl hydroperoxide (t-BOOH), onto rat spinal cord slice preparations. Field excitatory postsynaptic potentials (fEPSPs) evoked by dorsal root stimulations with either Aβ- or C-fiber intensity are recorded from the superficial dorsal horn. HFS significantly increases the slope of both Aβ- and C-fiber evoked fEPSPs, thus suggesting LTP development. The induction, not the maintenance, of HFS-induced LTP is blocked by a N-methyl-d-aspartate (NMDA) receptor antagonist, d-2-amino-5-phosphonopentanoic acid (d-AP5). Both the induction and maintenance of LTP of Aβ-fiber-evoked fEPSPs are inhibited by a ROS scavenger, either N-tert-butyl-α-phenylnitrone or 4-hydroxy-2,2,6,6-tetramethylpiperidine- N-oxyl. A ROS donor, t-BOOH-induced LTP is inhibited by N-tert-butyl-α-phenylnitrone but not by d-AP5. Furthermore, HFS-induced LTP and t-BOOH-induced LTP occlude each other. The data suggest that elevated ROS is a downstream event of NMDA receptor activation and an essential step for potentiation of synaptic excitability in the spinal dorsal horn.

The present study demonstrates many dynorphin (DYN)-immunoreactive fibers and presumed presynaptic terminals in rat lumbar ventral horn. The fibers and terminals seem to arise largely from DYN-containing intrinsic neurons in the dorsal horn. The majority of the presumed terminals closely surround a subpopulation of motoneurons that tend to be located in flexor motoneuron columns. Acute C fiber, but not A fiber, primary afferent stimulation depletes the ventral horn DYN immunostaining. We interpret these findings to indicate that the spinal DYN neurons are well positioned to serve both as modulators of nociceptive input and as interneurons in motor reflexes. We further hypothesize that the depletion of DYN-immunoreactivity that follows either acute C fiber stimulation or intense nociceptive stimuli may be the trigger for the upregulation in spinal cord DYN that occurs in models of chronic pain states.

Recently, an age-related increase in the number of dorsal root ganglion (DRG) cells was reported in adult rats. This suggests neurogenesis of adult primary afferent neurons, which would be an extremely important phenomenon if it occurred. Other evidence is not compatible with this idea, however, so the issue is not settled. The primary point of contention concerns the counts of DRG cells in relation to age. In our opinion, these disagreements arise, at least in part, because different counting methods give different results for the same material. Thus, any method for determining DRG cell numbers should be calibrated. We previously calibrated some of the common methods used to count DRG cells and found that an empirical method gave accurate cell counts. In the present study, we have used this method and asked whether an age-related increase in the number of lumbar DRG cells can be demonstrated in adult rats. Our data indicate that DRG cell numbers remain essentially constant from 3 to 22 months of age. Mos...

Although it has long been known that sodium channels play an important role in the generation of abnormal neuronal activity and neuropathic pain, it is only recently that we have begun to understand the subtypes of sodium channels which are particularly important in neuropathic pain. Many of the identified subtypes of sodium channels are localized in dorsal root ganglion (DRG) neurons. Based on their sensitivity to tetrodotoxin (TTX), these sodium channels are classified as TTX-sensitive (TTXs) or TTX-resistant (TTXr) subtypes. In in vitro electrophysiological experiments, ectopic discharges arising from DRG neurons with injured axons are blocked by TTX at doses that are too low to block TTXr subtypes. Furthermore, the same low doses of TTX applied to the DRG of the injured segment in neuropathic rats significantly reduce pain behaviours. These data suggest that TTXs subtypes of sodium channels are playing an important role in the generation of both ectopic discharges and neuropathic pain. Analysis of mRNA of the TTXs subtypes of sodium channels in the DRG after spinal nerve ligation showed that Nav1.3 (Type III) and Nax (NaG) are the only two subtypes that are up-regulated, suggesting their potentially important role in ectopic discharge and neuropathic pain generation.

Ankle sprain is a frequent injury in humans that results in pain, swelling and difficulty in walking on the affected side. Currently a suitable animal model resembling human ankle sprain is lacking. Here, we describe an animal ankle sprain model induced by ankle ligament injury (ALI) in rats. Cutting combinations of the lateral ankle ligament complex produced pain, edema and difficulty of weight bearing, thereby mimicking severe (grade III) ankle sprain in humans. Analgesic compounds, morphine and indomethacin, significantly reversed the reduced weight bearing, thus indicating that reduction of weight bearing is partially due to pain. The ALI model is a new ankle sprain model that may be useful for the study of ankle sprain pain mechanisms and treatments, as well as for the screening of new analgesic drugs.

We examined the possibility that peripheral nerve injury in the adult rat triggers sprouting of unmyelinated ventral root afferent fibers. Three to 5 months after the sciatic nerve was sectioned on one side in the adult rat, myelinated and unmyelinated fibers were counted at 3 sites along the length of the ventral root. A sciatic nerve lesion resulted in about a 3-fold increase in the number of unmyelinated fibers in the L5 ventral root. Our data suggest that a peripheral nerve lesion in the adult rat triggers sprouting of unmyelinated afferent fibers in the ventral root. No evidence was found that dorsal rhizotomy triggers sprouting of afferent fibers.

The present study shows that systemic capsaicin in adult rats results in a significant loss of axons in the subepidermal nerve plexus of the posterior leg but no loss of axons in the sural nerves of these same animals. These data are interpreted as indicating that the receptive part of the peripheral sensory axon is destroyed but that the cell body and most of the peripheral axon remains intact. Thus we suggest that the receptive part of the peripheral sensory axon is the most vulnerable part of the primary afferent neuron to capsaicin in these animals. These findings may explain the observation that adult rats treated with systemic capsaicin are deficient in their responses to certain painful stimuli but usually do not show obvious signs of primary afferent neuron death. We also suggest that as the dose of capsaicin is increased the whole neuron dies. It remains to be determined if the peripheral damage reported here is related to the striking loss of primary afferent markers in the dorsal horn that is also seen after this treatment.

Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, thus suggesting ROS involvement in central sensitization. To investigate ROS involvement in central sensitization, the effects of ROS scavengers and donors on pain behaviors were examined in mice. Capsaicin- induced hyperalgesia was used as a pain model since it has 2 distinctive pain components, primary and secondary hyperalgesia representing peripheral and central sensitization, respectively. Capsaicin (25 microg/5 microl) was injected intradermally into the left hind foot. Foot withdrawal frequencies in response to von Frey filament stimuli were measured and used as an indicator of mechanical hyperalgesia. The production of ROS was examined by using a ROS sensitive dye, MitoSox. Mice developed primary and secondary mechanical hyperalgesia after capsaicin injection. A systemic or intrathecal post-treatment with either phenyl-N-tert-butylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL), ROS scavengers, significantly reduced secondary hyperalgesia, but not primary hyperalgesia, in a dose-dependent manner. Pretreatment with ROS scavengers also significantly reduced the magnitude and duration of capsaicin-induced secondary hyperalgesia. On the other hand, intrathecal injection of tert-butylhydroperoxide (t-BOOH, 5 microl), a ROS donor, produced a transient hyperalgesia in a dose-dependent manner. The number of MitoSox positive dorsal horn neurons was increased significantly after capsaicin treatment. This study suggests that ROS mediates the development and maintenance of capsaicin-induced hyperalgesia in mice, mainly through central sensitization and that the elevation of spinal ROS is most likely due to increased production of mitochondrial superoxides in the dorsal horn neurons.

In a previous study, we showed that electroacupuncture (EA) applied to the SI-6 point on the contralateral forelimb produces long-lasting and powerful analgesia in pain caused by ankle sprain in a rat model. To investigate the underlying mechanism of EA analgesia, the present study tested the effects of various antagonists to known endogenous analgesic systems in this model. Ankle sprain was induced in anesthetized rats by overextending their right ankle with repeated forceful plantar flexion and inversion of the foot. When rats developed pain behaviors (a reduction in weight bearing of the affected hind limb), EA was applied to the SI-6 point on the contralateral forelimb for 30 minutes under halothane anesthesia. EA significantly improved the weight-bearing capacity of the affected hind limb for 2 hours, suggesting an analgesic effect. The alpha-adrenoceptor antagonist phentolamine (2 mg/kg, i.p. or 30 μg, i.t.) completely blocked the EA-induced analgesia, whereas naloxone (1 mg/kg, i.p.) and failed to block the effect. These results suggest that EA-induced analgesia is mediated by alpha-adrenoceptor mechanisms. Further experiments showed that intrathecal administration of yohimbine (10 μg), an α2-adrenergic antagonist, reduced the EA-induced analgesia in a dose-dependent manner, whereas terazosin (10 μg), an α1-adrenergic antagonist, did not produce any effect. These data suggest that the analgesic effect of EA in ankle sprain pain is, at least in part, mediated by spinal α2-adrenoceptor mechanisms.

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.

A number of good animal models have been developed in recent years that provide insights into the mechanisms of neuropathic pain. It now becomes evident that there are two separate peripheral components influencing neuropathic pain: one dependent on the hyperexcitability of axotomized dorsal root ganglion (DRG) neurons and the other independent of this hyperexcitability. The purpose of this review is to consider one of these components, the hyperexcitability of axotomized DRG neurons, as one of the important mechanisms underlying neuropathic pain. Several hours after nerve lesions, some axotomized DRG neurons become hyperexcitable and begin to show ongoing discharges that last many days or weeks. These ectopic discharges then enter the spinal cord and induce central sensitization, the underlying central mechanism for the generation of pain and allodynia. Although the exact causes of the development of hyperexcitability and ectopic discharges are not clear, various ion channels seem to play important roles, particularly sodium channels. In addition, important modulatory factors for ectopic discharges are purinergic and adrenergic components of the sympathetic nervous system. These findings suggest that manipulating sodium channels and/or adrenergic and purinergic receptors on axotomized DRG cells may give neuropathic pain sufferers some relief that is not available from present treatment regimens.

This study tests the hypothesis that central sensitization initiated by nociceptive input can be maintained by repeated brief innocuous peripheral inputs. Capsaicin was injected intradermally into the hind paw of adult rats. Three different types of daily cutaneous mechanical stimulations (vibration, soft brush, or pressure) were applied to the capsaicin-injected paw for a period of 2 weeks. Daily stimulation consisted of a 10-s stimulation repeated every 30s for 30 min. Foot withdrawal thresholds to von Frey stimuli applied to the paw were measured once a day for 4 weeks. The capsaicin-only group (control rats without daily stimulation) showed hyperalgesia lasting for 3 days. In contrast, hyperalgesia persisted for 2 weeks in the group that received vibration stimulation. Neither the soft brush nor the pressure group showed a significant difference in mechanical threshold from the control group (capsaicin only). The vibration-induced prolonged hyperalgesia was significantly reduced by systemic injection of ifenprodil, an NMDA-receptor antagonist, but it was not influenced by either an AMPA-receptor blocker or a reactive oxygen species (ROS) scavenger. Furthermore, a dorsal column lesion did not interfere with the prolongation of hyperalgesia. Data suggest that vibration-induced prolongation of hyperalgesia is mediated by spinal NMDA-receptors, and a similar mechanism may underlie some forms of chronic pain with no obvious causes, such as complex regional pain syndrome type 1 (CRPS-1).

Publisher Summary This chapter discusses evidence supporting the hypothesis that the alterations of one or two subtypes of voltage‐gated sodium channels (VGSCs) in axotomized afferents contribute to the generation and maintenance of neuropathic pain. VGSCs are the key element of excitable cells in the generation and transmission of electrical activity. VGSCs play a fundamentally important role in the excitability of almost all neurons in the central and peripheral nervous systems. They are located in the plasma membrane and mediate the influx of sodium ions into the cell in response to membrane depolarization. Sodium influx results in the generation of the action potential. Ten distinct pore‐forming subunits of VGSCs are identified in vertebrates and many of these are localized in dorsal root ganglion (DRG) neurons. Peripheral nerve injury result in neuropathic pain in which normally painful peripheral stimuli produce severe pain and normally nonpainful stimuli produce pain. Dynamic changes in VGSCs after nerve injury play an important role in generating ectopic discharges and spontaneous activity of sensory neurons.

Reactive oxygen species (ROS) are toxic agents that may be involved in various neurodegenerative diseases. Recent studies indicate that ROS are also involved in persistent pain through a spinal mechanism. Since the major source of ROS in neurons is mitochondria, mitochondrial ROS generation was examined in dorsal horn neurons of neuropathic rats. Neuropathic rats were produced by L5 spinal nerve ligation and mitochondrial ROS was detected by the mitochondrial marker, Mitotracker Red CM-H(2)XRos (MT-Red). Neurons were identified immunohistochemically for the neuronal marker NeuN. The number of MT-Red positive cells was increased 60-100% in the neuropathic dorsal horn. Approximately 75-85% of MT-Red positive cells were neurons. These data suggest that increased mitochondrial ROS in dorsal horn neurons may contribute to central sensitization in neuropathic rats.

Although it has long been known that sodium channels play an important role in the generation of abnormal neuronal activity and neuropathic pain, it is only recently that we have begun to understand the subtypes of sodium channels which are particularly important in neuropathic pain. Many of the identified subtypes of sodium channels are localized in dorsal root ganglion (DRG) neurons. Based on their sensitivity to tetrodotoxin (TTX), these sodium channels are classified as TTX-sensitive (TTXs) or TTX-resistant (TTXr) subtypes. In in vitro electrophysiological experiments, ectopic discharges arising from DRG neurons with injured axons are blocked by TTX at doses that are too low to block TTXr subtypes. Furthermore, the same low doses of TTX applied to the DRG of the injured segment in neuropathic rats significantly reduce pain behaviours. These data suggest that TTXs subtypes of sodium channels are playing an important role in the generation of both ectopic discharges and neuropathic pain. Analysis of mRNA of the TTXs subtypes of sodium channels in the DRG after spinal nerve ligation showed that Nav1.3 (Type III) and Nax (NaG) are the only two subtypes that are up-regulated, suggesting their potentially important role in ectopic discharge and neuropathic pain generation.

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 (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.

Primary cultures of neurons provide opportunities to study the cell biology of neurons under controlled conditions. Because differences exist in cellular properties among populations of neurons in the brain, survival requirements for neurons among these regions differ as well. This chapter outlines protocols for the preparation of primary cultures of spinal cord from 2-d-old neonatal rats. One protocol prepares cultures enriched in neurons and an alternative procedure prepares cultures enriched in non-neuronal cells. Comparison of biochemical data between these two culture preparations allows deductions of effects of treatments on neurons in the cultures. Limitations in interpretation of data obtained from cultured neurons are discussed.