Jigong Wang

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.

Persistent pain is dependent on spinal mitochondrial antioxidant levels.

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.

Mitochondrial Ca(2+) uptake is essential for synaptic plasticity in pain.

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+]c does not trigger synaptic plasticity directly but must first enter into mitochondria. Interfering with mitochondrial Ca2+ uptake during a [Ca2+]c increase 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+]c requires 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.

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.

Functional motoneurons develop from human neural stem cell transplants in adult rats.

We have shown previously that primed human fetal neural stem cells, after transplantation into rat spinal cords, differentiated into cholinergic motoneurons that sent axons to contact medial gastrocnemius myocytes. Here we demonstrate that (i) axons from the transplanted cells are cholinergic and myelinated, (ii) putative synapses form on transplanted somata and dendrites in the ventral horn, (iii) human fetal neural stem cells transplantation led to normal electromyograms from medial gastrocnemius muscles, and (iv) the gait of transplanted animals was much improved. Accumulatively, our data indicate that some transplanted human fetal neural stem cells in adult motoneuron-deficient ventral horns differentiate into relatively normal motoneurons that are integrated into spinal and peripheral circuitry. These findings are steps towards the long-term goal of providing stem cell transplants for motoneuron loss.

Effect of antioxidant treatment on spinal GABA neurons in a neuropathic pain model in the mouse

Summary This study demonstrates that neuropathic pain may be attributed to oxidative stress, which induces both a gamma‐aminobutyric acid neuron loss and dysfunction of surviving gamma‐aminobutyric acid neurons. Abstract 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 naïve 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 naïve 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 naïve 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.

Regulation of Wnt signaling by nociceptive input in animal models.

BackgroundCentral 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.ResultsFluorescent 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.ConclusionOur 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.

Superoxide signaling in pain is independent of nitric oxide signaling.

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 after spinal nerve ligation was attenuated by removing •O2− 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 •O2− but was reduced by a guanyl cyclase inhibitor. Superoxide-induced hyperalgesia was not affected by inhibiting NO• production but was suppressed by a protein kinase C inhibitor. The data suggest that NO• and •O2− operate independently to generate pain.

Phenyl N-t-butylnitrone, a reactive oxygen species scavenger, reduces zymosan-induced visceral pain in rats

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.

A surgical ankle sprain pain model in the rat: Effects of morphine and indomethacin

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.