Dionéia Araldi

Inflammation-induced decrease in voluntary wheel running in mice: a nonreflexive test for evaluating inflammatory pain and analgesia.

Summary Extent of voluntary locomotion by mice in activity wheels during peripheral inflammation is a simple, objective index of inflammatory pain, highly sensitive to analgesic/antiinflammatory treatment. Abstract Inflammatory pain impacts adversely on the quality of life of patients, often resulting in motor disabilities. Therefore, we studied the effect of peripheral inflammation induced by intraplantar administration of complete Freund’s adjuvant (CFA) in mice on a particular form of voluntary locomotion, wheel running, as an index of mobility impairment produced by pain. The distance traveled over 1 hour of free access to activity wheels decreased significantly in response to hind paw inflammation, peaking 24 hours after CFA administration. Recovery of voluntary wheel running by day 3 correlated with the ability to support weight on the inflamed limb. Inflammation‐induced mechanical hypersensitivity, measured with von Frey hairs, lasted considerably longer than the impaired voluntary wheel running and is not driving; therefore, the change in voluntary behavior. The CFA‐induced decrease in voluntary wheel running was dose‐dependently reversed by subcutaneous administration of antiinflammatory and analgesic drugs, including naproxen (10–80 mg/kg), ibuprofen (2.5–20 mg/kg), diclofenac (1.25–10 mg/kg), celecoxib (2.5–20 mg/kg), prednisolone (0.62–5 mg/kg), and morphine (0.06–0.5 mg/kg), all at much lower doses than reported in most rodent models. Furthermore, the doses that induced recovery in voluntary wheel running did not reduce CFA‐induced mechanical allodynia, indicating a greater sensitivity of the former as a surrogate measure of inflammatory pain. We conclude that monitoring changes in voluntary wheel running in mice during peripheral inflammation is a simple, observer‐independent objective measure of functional changes produced by inflammation, likely more aligned to the global level of pain than reflexive measures, and much more sensitive to analgesic drug effects.

On the mechanisms involved in antinociception induced by diphenyl diselenide

In this study, we described the local peripheral antinociceptive activity produced by diphenyl diselenide in the formalin test as compared to ebselen, an amply studied organoselenium compound. A second objective was to evaluate, the possible mechanisms underlying the antinociceptive effect caused by diphenyl diselenide. Administration of diphenyl diselenide or ebselen produced a significant antinociceptive local effect on the late phase (15-30min) of the formalin test. As well, diphenyl diselenide and ebselen injected in the contra lateral paw produced a significant decrease in licking time on the late phase (15-30min). The mechanisms underlying the analgesic action of diphenyl diselenide seem to be unlike the activation of opioid, dopaminergic D2, muscarinic cholinergic receptors or the interaction with α(1) and α(2) adrenoceptors. Furthermore, the effect of a 5-HT(3) receptor antagonist in abolishing the antinociception induced by diphenyl diselenide suggests the involvement of serotonergic pathways.

Repeated Mu-Opioid Exposure Induces a Novel Form of the Hyperalgesic Priming Model for Transition to Chronic Pain

The primary afferent nociceptor was used as a model system to study mechanisms of pain induced by chronic opioid administration. Repeated intradermal injection of the selective mu-opioid receptor (MOR) agonist DAMGO induced mechanical hyperalgesia and marked prolongation of prostaglandin E2 (PGE2) hyperalgesia, a key feature of hyperalgesic priming. However, in contrast to prior studies of priming induced by receptor-mediated (i.e., TNFα, NGF, or IL-6 receptor) or direct activation of protein kinase Cε (PKCε), the pronociceptive effects of PGE2 in DAMGO-treated rats demonstrated the following: (1) rapid induction (4 h compared with 3 d); (2) protein kinase A (PKA), rather than PKCε, dependence; (3) prolongation of hyperalgesia induced by an activator of PKA, 8-bromo cAMP; (4) failure to be reversed by a protein translation inhibitor; (5) priming in females as well as in males; and (6) lack of dependence on the isolectin B4-positive nociceptor. These studies demonstrate a novel form of hyperalgesic priming induced by repeated administration of an agonist at the Gi-protein-coupled MOR to the peripheral terminal of the nociceptor. SIGNIFICANCE STATEMENT The current study demonstrates the molecular mechanisms involved in the sensitization of nociceptors produced by repeated activation of mu-opioid receptors and contributes to our understanding of the painful condition observed in patients submitted to chronic use of opioids.

P2X3 and P2X2/3 receptors mediate mechanical hyperalgesia induced by bradykinin, but not by pro-inflammatory cytokines, PGE2 or dopamine

Activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP is essential to the development of inflammatory hyperalgesia. We have previously demonstrated that this essential role of P2X3 and P2X2/3 receptors in the development of mechanical hyperalgesia induced by the inflammatory agent carrageenan is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of tumor necrosis factor alpha (TNF-α) and by a direct sensitization of the primary afferent nociceptors. Therefore, in this study we asked whether activation of P2X3 and P2X2/3 receptors contribute to the mechanical hyperalgesia induced by the inflammatory mediators involved in carrageenan-induced mechanical hyperalgesia, such as bradykinin, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), chemokine-induced chemoattractant-1 (CINC-1), prostaglandin E₂ (PGE₂) and dopamine. Co-administration of the non-selective P2X3 receptor antagonist TNP-ATP or the selective P2X3 and P2X2/3 receptor antagonist A-317491 with bradykinin, but not with TNF-α, IL-1β, IL-6, CINC-1, PGE₂ or dopamine, prevented in a dose-dependent manner the mechanical hyperalgesia. We also verified whether the activation of P2X3 and P2X2/3 receptors by endogenous ATP contributes to bradykinin-induced mechanical hyperalgesia via neutrophil migration and/or cytokine release. Co-administration of TNP-ATP or A-317491 did not affect either neutrophil migration or the increased concentration of TNF-α, IL-1β, IL-6 and CINC-1 induced by bradykinin. These findings demonstrate that the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia by a mechanism that does not depend on neutrophil migration or cytokines release.

Inflammatory sensitization of nociceptors depends on activation of NMDA receptors in DRG satellite cells

Significance The present study provides evidence for a role of glutamate as a neuromodulator of the afferent nociceptive information. Our results show that the nociceptive impulse generated by an inflammatory event in the peripheral tissue is regulated in the dorsal root ganglia (DRG) by a system that involves satellite glial cells and glutamatergic NMDA receptors. To our knowledge, this work is one of the first demonstrations of the involvement of glutamate in a modulatory process in the DRG, a site where there are no synapses, in addition to its classical role as a neurotransmitter. The present study evaluated the role of N-methyl-d-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E2 (PGE2), which was significantly attenuated by injection of the NMDAR antagonist d(-)-2-amino-5-phosphonopentanoic acid (d-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE2-induced hyperalgesia in thepaw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE2-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE2-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 μL) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1β (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1β. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1β in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1+ cells of the DRG, significantly increased after carrageenan or IL-1β administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKCε expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 µg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1β in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.

Nanoassisted Laser Desorption-Ionization-MS Imaging of Tumors

The ability of nanoassisted laser desorption-ionization mass spectrometry (NALDI-MS) imaging to provide selective chemical monitoring with proper spatial distribution of lipid profiles from tumor tissues after plate imprinting has been tested. NALDI-MS imaging identified and mapped several potential lipid biomarkers in a murine model of melanoma tumor (inoculation of B16/F10 cells). It also confirmed that the in vivo treatment of tumor bearing mice with synthetic supplement containing phosphoethanolamine (PHO-S) promoted an accentuated decrease in relative abundance of the tumor biomarkers. NALDI-MS imaging is a matrix-free LDI protocol based on the selective imprinting of lipids in the NALDI plate followed by the removal of the tissue. It therefore provides good quality and selective chemical images with preservation of spatial distribution and less interference from tissue material. The test case described herein illustrates the potential of chemically selective NALDI-MS imaging for biomarker discovery.

Distinct Terminal and Cell Body Mechanisms in the Nociceptor Mediate Hyperalgesic Priming

Hyperalgesic priming, a form of neuroplasticity in nociceptors, is a model of the transition from acute to chronic pain in the rat, which involves signaling from the site of an acute tissue insult in the vicinity of the peripheral terminal of a nociceptor to its cell body that, in turn, induces a signal that travels back to the terminal to mediate a marked prolongation of prostaglandin E2-induced hyperalgesia. In the present experiments, we studied the underlying mechanisms in the cell body and compared them to the mechanisms in the nerve terminal. Injection of a cell-permeant cAMP analog, 8-bromo cAMP, into the dorsal root ganglion induced mechanical hyperalgesia and priming with an onset more rapid than when induced at the peripheral terminal. Priming induced by intraganglion 8-bromo cAMP was prevented by an oligodeoxynucleotide antisense to mRNA for a transcription factor, cAMP response element-binding protein (CREB), and by an inhibitor of importin, which is required for activated CREB to get into the nucleus. While peripheral administration of 8-bromo cAMP also produced hyperalgesia, it did not produce priming. Conversely, interventions administered in the vicinity of the peripheral terminal of the nociceptor that induces priming—PKCε activator, NGF, and TNF-α—when injected into the ganglion produce hyperalgesia but not priming. The protein translation inhibitor cordycepin, injected at the peripheral terminal but not into the ganglion, reverses priming induced at either the ganglion or peripheral terminal of the nociceptor. These data implicate different mechanisms in the soma and terminal in the transition to chronic pain.

Adenosine-A1 receptor agonist induced hyperalgesic priming type II.

Abstract We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt) induces a model of transition to chronic pain that we have termed type II hyperalgesic priming. Similar to type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, type II hyperalgesic priming differs from type I in being rapidly induced, protein kinase A (PKA), rather than PKC&egr; dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that, as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N6-cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced type II hyperalgesic priming. In this study, we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms, as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein &agr;i subunit activation, differently from DAMGO-induced type II priming, in which it depends on the &bgr;/&ggr; subunit. These data implicate a novel form of Gi-protein signaling pathway in the type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor.

Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming.

Abstract We have recently described a novel form of hyperalgesic priming (type II) induced by agonists at two clinically important Gi-protein–coupled receptors (Gi-GPCRs), mu-opioid and A1-adenosine. Like mu-opioids, the antimigraine triptans, which act at 5-HT1B/D Gi-GPCRs, have been implicated in pain chronification. We determined whether sumatriptan, a prototypical 5-HT1B/D agonist, produces type II priming. Characteristic of hyperalgesic priming, intradermal injection of sumatriptan (10 ng) induced a change in nociceptor function such that a subsequent injection of prostaglandin-E2 (PGE2) induces prolonged mechanical hyperalgesia. However, onset to priming was delayed 3 days, characteristic of type I priming. Also characteristic of type I priming, a protein kinase C&egr;, but not a protein kinase A inhibitor attenuated the prolongation phase of PGE2 hyperalgesia. The prolongation of PGE2 hyperalgesia was also permanently reversed by intradermal injection of cordycepin, a protein translation inhibitor. Also, hyperalgesic priming did not occur in animals pretreated with pertussis toxin or isolectin B4–positive nociceptor toxin, IB4–saporin. Finally, as observed for other agonists that induce type I priming, sumatriptan did not induce priming in female rats. The prolongation of PGE2 hyperalgesia induced by sumatriptan was partially prevented by coinjection of antagonists for the 5-HT1B and 5-HT1D, but not 5-HT7, serotonin receptors and completely prevented by coadministration of a combination of the 5-HT1B and 5-HT1D antagonists. Moreover, the injection of selective agonists, for 5-HT1B and 5-HT1D receptors, also induced hyperalgesic priming. Our results suggest that sumatriptan, which signals through Gi-GPCRs, induces type I hyperalgesic priming, unlike agonists at other Gi-GPCRs, which induce type II priming.