Expression of orphanin FQ/nociceptin and its receptor in rat peripheral ganglia and spinal cord

Expression of the neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, the opioid receptor-like receptor (ORL1), have been found to have a wide distribution in the central nervous system, and in brain areas involved in sensory perception in particular. The effects of OFQ/N on, e.g., sensory transmission are very complex, and a modulatory effect on pain perception has been suggested. We therefore wanted to investigate the distribution of OFQ/N and ORL1 in the spinal cord and DRG, and also in SCG and some other peripheral tissues. The methods used were in situ hybridization, immunohistochemistry and ligand binding. We found that OFQ/N and ORL1 mRNA are expressed in DRG; primarily in small and large neurons, respectively. In spinal cord, mRNA for OFQ/N and ORL1 is expressed in neurons in laminae I, II and X, and in ventral horn neurons. Further, immunoreactivity for OFQ/N is observed in fibers and neurons in the superficial laminae of the dorsal horn and around the central canal, and also in neurons in the ventral horn of the spinal cord. Receptor ligand binding to the spinal cord grey matter is demonstrated, primarily concentrated to the dorsal horn and around the central canal, and also to medium and large size DRG neurons. These findings on the morphological distribution pattern of OFQ/N and ORL1 at the cellular level may support the notion that OFQ/N is involved in modulating pain transmission. Further, expression of OFQ/N and ORL1 mRNA was also found in SCG, whereas expression was undetectable in skin.     

Two major distinct subpopulations of neurokinin-3 receptor-expressing neurons in the superficial dorsal horn of the rat spinal cord

This study was designed to provide evidence for elucidating the mechanisms of neurokinin-3 receptor (NK3) in spinal pain modulation. First, colocalization of NK3 with the micro -opioid receptor (MOR1) was studied in the spinal dorsal horn of the rat. Confocal microscopy showed that about 44% of NK3-expressing neurons in laminae I and II were immunoreactive for MOR1, which corresponded to about 93% of the total population of MOR1-containing neurons in these laminae. Second, the relationship between NK3/MOR1-coexpressing neurons and those that express nitric oxide synthase (NOS) was examined by using a triple immunofluorescent staining method. About 37% of NK3-immunoreactive neurons were also NOS-immunoreactive, which constituted about 82% of NOS-immunoreacitve neurons in the superficial laminae. However, no triple-labelled neurons were detected. The present results indicate that there are two major distinct subpopulations of NK3-expressing neurons in the superficial dorsal horn, which suggests that the involvement of NK3 receptor in spinal nociception could be mediated by two distinct mechanisms, i.e. opioid and nitric oxide.     

Electroacupuncture (EA) modulates the expression of NMDA receptors in primary sensory neurons in relation to hyperalgesia in rats

N-methyl-D-aspartate (NMDA) receptor on the central terminals of the dorsal root ganglion (DRG) appears to be playing an important role in the development of central sensitization related to persistent inflammatory pain. Acupuncture analgesia has been confirmed by numerous clinical observations and experimental studies to be a useful treatment to release different kinds of pains, including inflammatory pain and hyperalgesia. However, the underlying mechanisms of the analgesic effect of acupuncture are not fully understood. In the present study, using a rat model of inflammatory pain induced by complete Freund's adjuvant (CFA), we observed the effect of electroacupuncture (EA) on animal behavior with regard to pain and the expression of a subunit of NMDA receptor (NR1) and isolectin B4 (IB4) in the neurons of the lumbar DRG. Intraplantar injection of 50 microl CFA resulted in considerable changes in thermal hyperalgesia, edema of the hind paw and "foot-bend" score, beginning 5 h post-injection and persisting for a few days, after which a gradual recovery occurred. The changes were attenuated by EA treatment received on the ipsilateral "Huan Tiao" and "Yang Ling Quan" once a day from the first day post-injection of CFA. Using an immunofluorescence double staining, we found that the number of double-labeled cells to the total number of the IB4 and NR1-labeled neurons increased significantly on days 3 and 7 after CFA injection. The change was attenuated by EA treatment. These results suggest that EA affects the progress of experimental inflammatory pain by modulating the expression of NMDA receptors in primary sensory neurons, in particular, IB4-positive small neurons.     

The involvement of spinal bovine adrenal medulla 22-like peptide, the proenkephalin derivative, in modulation of nociceptive processing

Bovine adrenal medulla 22 (BAM22), one of the cleavage products of proenkephalin A, possesses high affinity for opioid receptors and sensory neuron‐specific receptor (SNSR). The present study was designed to examine the expression of BAM22 in the spinal cord and dorsal root ganglion (DRG) of naive rats as well as in a model of inflammation. BAM22‐like immunoreactivity (BAM22‐IR) was expressed in fibers in the spinal cord, with high density seen in lamina I in naïve rats. The expression of BAM22‐IR in the superficial laminae was greatly reduced following dorsal rhizotomy. BAM22‐IR was also located in 19% of DRG cells, mainly in the small‐ and medium‐sized subpopulations. Following injection of complete Freund's adjuvant (CFA) in the hindpaw, the expression of BAM22‐IR in the superficial laminae of the spinal cord and small‐sized DRG neurons on the ipsilateral side was markedly increased. Double labeling showed that the Fos‐positive nucleus was surrounded by BAM22‐IR cytoplasm in the spinal dorsal horn neurons or closely associated with BAM22‐IR fibers in the superficial laminae. Furthermore, CFA‐induced mechanical allodynia in the inflamed paw was potentiated by intrathecal administration of anti‐BAM22 antibody. Together, these results demonstrate for the first time that BAM22‐like peptide is mainly located in the superficial laminae of the spinal cord and mostly originates from nociceptive DRG neurons. BAM22 could thus act as a ligand for presynaptic opioid receptors and SNSR. Our study also provides evidence suggesting that BAM22 plays a role in the modulation of nociceptive processing at the spinal level under normal and inflammatory conditions.     

Peripheral purinergic receptor modulation influences the trigeminal ganglia nitroxidergic system in an experimental murine model of inflammatory orofacial pain

ATP plays an important role as an endogenous pain mediator generating and/or modulating pain signaling from the periphery to the central nervous system. The aim of this study was to analyze the role of peripheral purinergic receptors in modulation of the nitroxidergic system at a trigeminal ganglia level by monitoring changes in nitric oxide synthase isoforms. We also evaluated Fos‐positive neurons in brainstem (spinal trigeminal nucleus) and pain‐related behavior. We found that local administration of the P2 purinergic receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) decreased face‐rubbing activity, nitric oxide synthase isoform expression in trigeminal ganglia, and Fos expression in spinal trigeminal nucleus after subcutaneous injection of formalin. These results suggest a role for peripheral P2 purinergic receptors in orofacial pain transmission through modulation of the nitroxidergic system. © 2010 Wiley‐Liss, Inc.     

Involvement of endogenous opioid systems in nociceptin-induced spinal antinociception in rats

The present study investigates the involvement of opioid receptors in the antinociceptive effects of nociceptin in the spinal cord of the rat. Intrathecal administrations of 5 and 10 nmol of nociceptin significantly increase the withdraw response latencies to noxious thermal and mechanical stimulations. This nociceptin-induced antinociceptive effect is significantly attenuated by intrathecal injection of (Nphe(1))nociceptin(1-13)-NH(2), a selective antagonist of the nociceptin receptor (opioid receptor-like receptor ORL1), indicating an ORL1 receptor-mediated mechanism. This antinociceptive effect is also significantly attenuated by intrathecal injections of naloxone (a nonselective opioid receptor antagonist), naltrindole (a selective delta-opioid receptor antagonist), and beta-funaltrexamine (a selective mu-opioid receptor antagonist) in a dose-dependent manner, but not by the selective kappa-opioid receptor antagonist norbinaltorphimine. Since it is unlikely that nociceptin acts by direct binding to opioid receptors, these results suggest a possible interaction between the nociceptin/ORL1 and opioid systems in the dorsal horn of the rat spinal cord.     

Ligustilide attenuates inflammatory pain via inhibition of NFκB‐mediated chemokines production in spinal astrocytes

Ligustilide (LIG) is a major component of Radix Angelica Sinensis, and reportedly has neuroprotective and anti‐inflammatory effects. Recent studies have demonstrated that spinal astrocyte‐mediated neuroinflammation plays an important role in the pathogenesis of chronic pain. Here we investigated the anti‐nociceptive effect of systemic treatment with LIG on chronic inflammatory pain and explored possible mechanisms. Unilateral hindpaw injection of complete Freund's adjuvant (CFA) induced persistent pain hypersensitivity. Repeated daily intravenous treatment with LIG, either before or after CFA injection, attenuated CFA‐induced thermal hyperalgesia and mechanical allodynia. The same treatment also inhibited CFA‐induced keratinocyte‐derived chemokine (KC) and monocyte chemoattractant protein‐1 (MCP‐1) mRNA and protein increases in astrocytes of the spinal cord. In vitro study showed LIG dose‐dependently reduced lipopolysaccharide (LPS)‐induced upregulation of KC and MCP‐1 mRNA in astrocyte cultures. Interestingly, LIG treatment did not affect CFA‐ or LPS‐induced glial fibrillary acidic protein upregulation, but did inhibit CFA‐induced phosphorylated nuclear factor‐κB (p‐NFκB) upregulation in spinal astrocytes. Furthermore, intrathecal injection of NFκB inhibitor attenuated CFA‐induced pain hypersensitivity and upregulation of KC and MCP‐1 in the spinal cord. Finally, single intravenous injection of LIG attenuated intrathecal injection of LPS‐induced mechanical allodynia. The same treatment also decreased LPS‐induced NFκB activation and KC and MCP‐1 upregulation in the spinal cord. These data indicate that LIG attenuates chronic inflammatory pain potentially via inhibiting NFκB‐mediated chemokines production in spinal astrocytes. These results provide direct evidence of the anti‐nociceptive and anti‐inflammatory effects of LIG, suggesting a new application of LIG for the treatment of chronic inflammatory pain.     

Cutaneous inflammation regulates THIK1 expression in small C-like nociceptor dorsal root ganglion neurons

Tandem pore-domain Halothane Inhibited K⁺ channel (THIK1) is a two-pore-domain potassium channel (K2P) present in dorsal root ganglia (DRG). We previously demonstrated that THIK1 mRNA levels in the DRG dropped ipsilaterally 1 day after CFA-induced cutaneous inflammation (CFA1). In this study we aimed to identify the currently unknown DRG subpopulations expressing THIK1, and to investigate the relationship between the channel and both inflammatory and spontaneous pain in normal rats. Using a combination of immunohistochemistry, western blotting and behavioural tests, we found that all small neurons and large groups of medium and large DRG neurons express THIK1. Myelinated and unmyelinated fibers, nerve endings in the skin and lamina I and II of the spinal cord also express the channel. THIK1 staining co-localizes with IB4-binding and trkA suggesting that the channel is expressed by nociceptors. At CFA1, both cytoplasmic and edge (membrane-associated) THIK1 staining were significantly reduced only in small neurons ipsilaterally compared to normal. At 4 days after inflammation (CFA4), edge THIK1 staining levels in small neurons decreased bilaterally compared to normal. Medium and large size DRG neurons showed no change in THIK1 expression either at CFA1 or CFA4. Ipsilateral (but not contralateral) mean %intensities of THIK1 in small neurons at CFA1 correlated strongly negatively with spontaneous foot lifting (SFL) duration (a marker of spontaneous pain). Thus, nociceptors express THIK1 that can be regulated by cutaneous inflammation. Finally, in vivo siRNA knockdown of THIK1 resulted in longer SFL duration than siRNA scramble-treated rats. Taken together our evidence suggests a potential involvement for THIK1 in pain processing following inflammation.     

TRPM2 contributes to inflammatory and neuropathic pain through the aggravation of pronociceptive inflammatory responses in mice

Accumulating evidence suggests that neuroimmune interactions contribute to pathological pain. Transient receptor potential melastatin 2 (TRPM2) is a nonselective Ca2?-permeable cation channel that acts as a sensor for reactive oxygen species. TRPM2 is expressed abundantly in immune cells and is important in inflammatory processes. The results of the present study show that TRPM2 plays a crucial role in inflammatory and neuropathic pain. While wild-type and TRPM2 knock-out mice showed no difference in their basal sensitivity to mechanical and thermal stimulation, nocifensive behaviors in the formalin test were reduced in TRPM2 knock-out mice. In carrageenan-induced inflammatory pain and sciatic nerve injury-induced neuropathic pain models, mechanical allodynia and thermal hyperalgesia were attenuated in TRPM2 knock-out mice. Carrageenan-induced inflammation and sciatic nerve injury increased the expression of TRPM2 mRNA in the inflamed paw and around the injured sciatic nerve, respectively. TRPM2 deficiency diminished the infiltration of neutrophils and the production of chemokine (C-X-C motif) ligand-2 (CXCL2), a major chemokine that recruits neutrophils, but did not alter the recruitment of F4/80-positive macrophages in the inflamed paw or around the injured sciatic nerve. Microglial activation after nerve injury was suppressed in the spinal cord of TRPM2 knock-out mice. Furthermore, CXCL2 production and inducible nitric oxide synthase induction were diminished in cultured macrophages and microglia derived from TRPM2 knock-out mice. Together, these results suggest that TRPM2 expressed in macrophages and microglia aggravates peripheral and spinal pronociceptive inflammatory responses and contributes to the pathogenesis of inflammatory and neuropathic pain.     

Spinal IL-36γ/IL-36R participates in the maintenance of chronic inflammatory pain through astroglial JNK pathway

Emerging evidence indicates that spinal neuroinflammation contributes to the maintenance of chronic inflammatory pain. IL-36, as a novel member of the interleukin (IL)-1 super-family cytokines, plays an important role in inflammatory responses. The present study aimed to investigate the role of spinal IL-36 and IL-36 receptor (IL-36R) signaling in the pathology of chronic inflammatory pain. IL-36γ and IL-36R, but not IL-36α and IL-36β, were persistently upregulated in the spinal cord of mice with intraplantar injections of complete Freund's adjuvant (CFA). Intrathecal administration of both IL-36R antagonist (IL-36Ra) and IL-36γ siRNA significantly attenuated CFA-induced chronic inflammatory pain behaviors. Furthermore, CFA-induced IL-36γ expression was mainly observed in spinal neurons whereas IL-36R was primarily expressed in spinal astrocytes. Additionally, the intrathecal injection of IL-36γ was sufficient to induce pain hypersensitivity and astrocyte activation in naive mice, and these effects could be inhibited by blocking c-Jun N-terminal kinase (JNK) phosphorylation. In vitro experiments also demonstrated that the IL-36γ could induce astrocytic JNK activation and inflammatory cytokines release, which was mediated by IL-36R. Finally, intrathecal injection of IL-36γ-activated astrocytes in a pJNK-dependent manner induced mechanical allodynia and thermal hyperalgesia in naive mice. Collectively, these findings reveal that the neuronal/astrocytic interaction in the spinal cord by which neuronally produced IL-36γ activates astrocytes via IL-36R-mediated JNK pathway is crucial for the maintenance of chronic inflammatory pain. Thus, IL-36γ/IL-36R-mediated astrocyte signaling may be a suitable therapeutic target for chronic inflammatory pain.     

Mu opioid receptor-containing neurons mediate electroacupuncture-produced anti-hyperalgesia in rats with hind paw inflammation

Previous studies showed that electroacupuncture (EA) significantly attenuates inflammatory hyperalgesia in a complete Freund's adjuvant (CFA)-induced inflammatory pain rat model. The present study demonstrates that pretreatment with Derm-sap, a selective toxin for neurons that contain mu opioid receptor (MOR), specifically decreases MOR and blocks EA anti-hyperalgesia. These data suggest that spinal MOR-containing neurons are involved in the processes by which EA produces anti-hyperalgesia.     

Distribution of immunoreactivity for the NK1 receptor on different subpopulations of sympathetic preganglionic neurons in the rat

The distribuktion of immunoreactivity to the recptor for substance P, the neurokinin 1 (NK1) receptor, was ezamined in preganglionic sympathetic neurons of the rat by usingimmunohistochemistry and retroggrade neuronal tracing abour one-third of all sympathetic preganglionic neurons were NK1 receptor immunoreactiv, and most of the NK1 receptor immunoractive neurons were also nitric oxide synthase immunoreactive the proportions ofsympathetic preganglionic neurons projecting to the superior and inferior mesenteric ganglia, addrenal glnd, and lumbar sympathetic hain which werre nK1 rdeptor-immunoreactive were determined. Most (89%) of the preganglionic neurons projecting to the addrenal glands were NK1 rceptor immunoreactive few (17%) of the preganglionic neurons projecting to the L5 sympathetic hain ganglion were immunoreactive for the receptor, while preganglionic neurons projecting tothe prevertebral ganglia were NK1 receptor immunoractive at intermediate frequencies (61–64%). Thus, substance P acting on NK1 receptors in likely to be importnt inthe pregnglionic pathways to the addrenal medulla and viscera via the preveertebral ganglia, but the NK1 receptor withthe enzyme nitric oxide synthase was also examined. The co-localisation of NK1 receptor-immunoreactive neurons were also nitric oxide synthase immunoreactiv. Thus NK1 receptors occur onpreganglionic neurons over many spinal segments and in a range of prganglionic pathways, as well as in a range of combinations with nitric oxide shnthase. The heterogeneity ofjpreganglionic neurons showing NK1 receeptor immunoreactivity may reglect the involvement of NK1-mediated trnsmkssion in a variety of functional pathways, most notably thepreganglionic projections to the adrenal medulla and to the viscera. © 1996 Wiley-Liss, Inc.     

Inhibitory effects of CB1 and CB2 receptor agonists on responses of DRG neurons and dorsal horn neurons in neuropathic rats

Cannabinoid 2 (CB2) receptor mediated antinociception and increased levels of spinal CB2 receptor mRNA are reported in neuropathic Sprague-Dawley rats. The aim of this study was to provide functional evidence for a role of peripheral, vs. spinal, CB2 and cannabinoid 1 (CB1) receptors in neuropathic rats. Effects of the CB2 receptor agonist, JWH-133, and the CB1 receptor agonist, arachidonyl-2-chloroethylamide (ACEA), on primary afferent fibres were determined by calcium imaging studies of adult dorsal root ganglion (DRG) neurons taken from neuropathic and sham-operated rats. Capsaicin (100 nm) increased [Ca2+]i in DRG neurons from sham and neuropathic rats. JWH-133 (3 microm) or ACEA (1 microm) significantly (P<0.001) attenuated capsaicin-evoked calcium responses in DRG neurons in neuropathic and sham-operated rats. The CB2 receptor antagonist, SR144528, (1 microm) significantly inhibited the effects of JWH-133. Effects of ACEA were significantly inhibited by the CB1 receptor antagonist SR141716A (1 microm). In vivo experiments evaluated the effects of spinal administration of JWH-133 (8-486 ng/50 microL) and ACEA (0.005-500 ng/50 microL) on mechanically evoked responses of neuropathic and sham-operated rats. Spinal JWH-133 attenuated mechanically evoked responses of spinal neurons in neuropathic, but not sham-operated rats. These inhibitory effects were blocked by SR144528 (0.001 microg/50 microL). Spinal ACEA inhibited mechanically evoked responses of neuropathic and sham-operated rats, these effects were blocked by SR141716A (0.01 microg/50 microL). Our data provide evidence for a functional role of CB2, as well as CB1 receptors on DRG neurons in sham and neuropathic rats. At the level of the spinal cord, CB2 receptors have inhibitory effects in neuropathic, but not sham-operated rats suggesting that spinal CB2 may be an important analgesic target.     

Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia

Spinal proinflammatory cytokines are powerful pain-enhancing signals that contribute to pain following peripheral nerve injury (neuropathic pain). Recently, one proinflammatory cytokine, interleukin-1, was also implicated in the loss of analgesia upon repeated morphine exposure (tolerance). In contrast to prior literature, we demonstrate that the action of several spinal proinflammatory cytokines oppose systemic and intrathecal opioid analgesia, causing reduced pain suppression. In vitro morphine exposure of lumbar dorsal spinal cord caused significant increases in proinflammatory cytokine and chemokine release. Opposition of analgesia by proinflammatory cytokines is rapid, occurring 5 min after intrathecal (perispinal) opioid administration. We document that opposition of analgesia by proinflammatory cytokines cannot be accounted for by an alteration in spinal morphine concentrations. The acute anti-analgesic effects of proinflammatory cytokines occur in a p38 mitogen-activated protein kinase and nitric oxide dependent fashion. Chronic intrathecal morphine or methadone significantly increased spinal glial activation (toll-like receptor 4 mRNA and protein) and the expression of multiple chemokines and cytokines, combined with development of analgesic tolerance and pain enhancement (hyperalgesia, allodynia). Statistical analysis demonstrated that a cluster of cytokines and chemokines was linked with pain-related behavioral changes. Moreover, blockade of spinal proinflammatory cytokines during a stringent morphine regimen previously associated with altered neuronal function also attenuated enhanced pain, supportive that proinflammatory cytokines are importantly involved in tolerance induced by such regimens. These data implicate multiple opioid-induced spinal proinflammatory cytokines in opposing both acute and chronic opioid analgesia, and provide a novel mechanism for the opposition of acute opioid analgesia.     

Change of vanilloid receptor 1 expression in dorsal root ganglion and spinal dorsal horn during inflammatory nociception induced by complete Freund's adjuvant in rats

The present study aimed to systematically observe the change of vanilloid receptor 1 (VR1) during inflammatory nociception induced by intraplantar injection of complete Freund's adjuvant (CFA) into the left hind paw in rats. Hot plate latency (HPL) was used to evaluate resulting thermal hyperalgesia and immunohistochemistry to observe VR1 expression in dorsal root ganglion and spinal cord dorsal horn. Results showed that HPL decreased from day 1 to day 28 after CFA injection, with shortest at day 14. VR1 expression correspondingly increased from day 1 to day 21 with peak at day 14, and returning to the control level at day 28. A shift of VRI expression from small to medium DRG neurons over the observation period was seen. These results suggest that VR1 could play an important role in the early stage, but not the late stage, of CFA inflammatory nociception.     

Chronic selective activation of excitatory opioid receptor functions in sensory neurons results in opioid 'dependence' without tolerance.

We previously showed that mouse sensory dorsal root ganglion (DRG) neurons chronically exposed to 1 microM D-ala2-D-leu5-enkephalin (DADLE) or morphine for > 2-3 days in culture become tolerant to the usual opioid inhibitory receptor-mediated effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD), and supersensitive to opioid excitatory APD-prolonging effects elicited by low opioid concentrations. Whereas nanomolar concentrations of dynorphin(1-13) or morphine are generally required to prolong the APD of naive DRG neurons (by activating excitatory opioid receptors), femtomolar levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine prevent both excitatory and inhibitory opioid effects but do not alter the APD of native DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the chronic opioid-treated cells. In the present study, chronic exposure of DRG neurons to 1 microM DADLE together with cholera toxin-B subunit (which selectively blocks GM1 ganglioside-regulated opioid excitatory, but not inhibitory, receptor functions) prevented the development of opioid excitatory supersensitivity and markedly attenuated tolerance to opioid inhibitory effects. Conversely, sustained exposure of DRG neurons to 1 nM DADLE, which selectively activates excitatory opioid receptor functions, resulted in characteristic opioid excitatory supersensitivity but no tolerance. These results suggest that 'dependence'-like properties can be induced in chronic opioid-treated sensory neurons in the absence of tolerance. On the other hand, development of some components of tolerance in these cells may require sustained activation of both excitatory, as well as inhibitory, opioid receptor functions.     

Effect of Peripheral Axotomy on Expression of Neuropeptide Y Receptor mRNA in Rat Lumbar Dorsal Root Ganglia

Using in situ hybridization, the expression of the mRNA for a neuropeptide Y (NPY) receptor, was studied in lumbar (L) 4 and 5 dorsal root ganglia (DRGs) of normal rats and at various intervals after unilateral sciatic nerve transection. Twenty percent of all normal DRG neurons were NPY receptor mRNA-positive, and the majority of these neurons were of the small type, with only a few labelled medium-sized and large neurons. In L5 normal ganglia NPY receptor mRNA colocalized with substance P, calcitonin gene-related peptide and galanin mRNAs in small neurons, but not in medium-sized or large neurons containing these peptides. NPY receptor mRNA was not observed in somatostatin or nitric oxide synthase mRNA-positive neurons. Sciatic nerve transection induced a marked decrease in NPY receptor mRNA levels. However, in parallel there was a transient increase in the number of NPY receptor mRNA-positive small neuron profiles, but the intensity of labelling was mostly very low, although a few strongly labelled, small neuron profiles were also encountered. In addition, axotomy caused a marked increase in the number of NPY receptor mRNA-positive large neuron profiles in the ipsilateral DRGs, and they constituted 15–20% of counted DRG neuron profiles and 45–65% of counted large neuron profiles, 7–28 days after axotomy. In L5 DRGs, ipsilateral to the axotomy, NPY receptor mRNA colocalized with NPY mRNA in many large and some medium-sized neuron profiles, with galanin mRNA in some small, medium-sized and large neuron profiles and with vasoactive intestinal polypeptide mRNA in some small and medium-sized neuron profiles and a few large profiles. Occasionally, NPY receptor mRNA was observed in nitric oxide synthase mRNA-positive small neurons. In the dorsal horn, NPY receptor mRNA-positive small neurons were concentrated in lamina II at L4 and L5 levels, and were scattered in deeper laminae. No marked changes were observed ipsilateral to the axotomy. No NPY receptor mRNA-positive cells were found in the normal rat gracile nucleus, or in this nucleus after axotomy. These results show that a NPY receptor may be a prejunctional receptor in primary afferent neurons and play a role in the modulation of somatosensatory information, both in normal and lesioned primary afferent DRG cells. However, axotomy induced a distinct shift in NPY receptor mRNA expression from small to large neurons, indicating that sensitivity to NPY is switched from one modality to another. Thus, not only several sensory neuropeptides, as shown in previous studies, but at least also one of the peptide receptors change their expression dramatically in response to axotomy, suggesting complex adaptive responses.     

G2A as a Threshold Regulator of Inflammatory Hyperalgesia Modulates Chronic Hyperalgesia

Tissue injury, pathogen infection, and diseases are often accompanied by inflammation to release mediators that sensitize nociceptors and further recruit immune cells, which can lead to chronic hyperalgesia and inflammation. Tissue acidosis, occurring at the inflammatory site, is a major factor contributing to pain and hyperalgesia. The receptor G2 accumulation (G2A), expressed in neurons and immune cells, responds to protons or oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid produced by injured cells or oxidative stresses. We previously found increased G2A expression in mouse dorsal root ganglia (DRG) at 90 min after complete Freund’s adjuvant (CFA)-induced inflammatory pain, but whether G2A is involved in the inflammation or hyperalgesia remained unclear. In this study, we overexpressed or knocked-down G2A gene expression in DRG to explore the roles of G2A. G2A overexpression reduced the infiltration of acute immune cells (granulocytes) and attenuated hyperalgesia at 90 to 240 min after CFA injection. G2A knockdown increased the number of immune cells before CFA injection and prolonged the inflammatory hyperalgesia after CFA injection. G2A may serve as a threshold regulator in neurons to attenuate the initial nociceptive and inflammatory signals, modulating the chronic state of hyperalgesia.     

Phylogenetic changes in the expression of delta opioid receptors in spinal cord and dorsal root ganglia

To assess the validity of rodent models for investigating the role of delta opioid receptors (DOR) in analgesia, the distribution of DOR binding and mRNA were compared between rodent and primate spinal cord and dorsal root ganglia (DRG), using receptor autoradiography and in situ hybridization, respectively. In mouse and rat spinal cord, [(125)I]-deltorphin-labeled DOR binding sites were detected throughout the gray matter. In contrast, in primate and particularly in human spinal cord, DOR binding was mainly present in laminae I-II, with little to no binding in deeper layers. Accordingly, in rodent spinal cord, DOR mRNA was expressed by a large number of neurons distributed throughout the ventral and dorsal horns, whereas in the primate, DOR expression was significantly lower, as evidenced by a moderate number of labeled cells throughout the gray matter in monkey and by only few labeled cells in human, mainly in Clarke's column and lamina IX. Major species differences in DOR expression were also observed in primary afferent cells bodies. In rat DRG, intense DOR mRNA hybridization was primarily observed over large ganglion cells immunopositive for neurofilament 200. In contrast, in monkey and human DRG, DOR mRNA was primarily detected over small and medium-sized ganglion cells. These results demonstrate major differences in the expression and distribution of DOR in the spinal cord and DRG between mammalian species. Specifically, they point to a progressive specialization of DOR toward the regulation of primary somatosensory, namely nociceptive, inputs during phylogeny and suggest that the effects of DOR agonists in rodents may not be entirely predictive of their action in humans.