Carlos Amilcar Parada

Transient Receptor Potential Vanilloid 4 Is Essential in Chemotherapy-Induced Neuropathic Pain in the Rat

The development of treatments for neuropathic pain has been hindered by our limited understanding of the basic mechanisms underlying abnormalities in nociceptor hyperexcitability. We recently showed that the polymodal receptor transient receptor potential vanilloid 4 (TRPV4), a member of the transient receptor potential (TRP) family of ion channels, may play a role in inflammatory pain (Alessandri-Haber et al., 2003). The present study tested whether TRVP4 also contributes to neuropathic pain, using a rat model of Taxol-induced painful peripheral neuropathy. Taxol is the most widely used drug for the treatment of a variety of tumor types, but the dose of Taxol that can be tolerated is limited by the development of a small-fiber painful peripheral neuropathy. We found that Taxol treatment enhanced the nociceptive behavioral responses to both mechanical and hypotonic stimulation of the hind paw. Spinal administration of antisense oligodeoxynucleotides to TRPV4, which reduced the expression of TRPV4 in sensory nerve, abolished Taxol-induced mechanical hyperalgesia and attenuated hypotonic hyperalgesia by 42%. The enhancement of osmotic nociception involves sensitization of osmotransduction in primary afferents because osmotransduction was enhanced in cultured sensory neurons isolated from Taxol-treated rats. Taxol-induced TRPV4-mediated hyperalgesia and the enhanced osmotransduction in cultured nociceptors were dependent on integrin/Src tyrosine kinase signaling. These results suggest that TRPV4 plays a crucial role in a painful peripheral neuropathy, making it a very promising target for the development of a novel class of analgesics.

Tumor necrosis factor receptor type‐1 in sensory neurons contributes to induction of chronic enhancement of inflammatory hyperalgesia in rat

Carrageenan‐induced inflammatory pain lasting hours to days produces a protein kinase C epsilon (PKCɛ)‐dependent ‘primed’ state lasting several weeks, during which time injection of prostaglandin E2 induces hyperalgesia which is markedly enhanced and prolonged compared to PGE2‐induced hyperalgesia in normal ‘unprimed’ rats. In the present study, we demonstrate that while inhibition of prostaglandin synthesis and antagonism of β2‐adrenergic receptors markedly attenuated the hyperalgesia induced by carrageenan, these interventions did not affect hyperalgesic priming. Tumor necrosis factor‐α (rat recombinant; rrTNFα), another mediator of carrageenan‐induced inflammation, alone produced hyperalgesia and priming, which were attenuated and prevented, respectively, by intrathecal administration of antisense to PKCɛ. Inhibition of TNFα with thalidomide or a rat polyclonal anti‐TNFα antibody attenuated carrageenan‐induced hyperalgesia and prevented priming. Intrathecal administration of antisense to tumour necrosis factor receptor type‐1 (TNFR1) reduced the level of TNFR1 transported toward the peripheral terminals of sensory neurons, and attenuated both carrageenan‐ and rrTNFα‐induced priming. Acute hyperalgesia induced by carrageenan or rrTNFα remained intact in animals treated with TNFR1 antisense. Our results demonstrate that the generation of the primed state does not require production of hyperalgesia and that TNFα, which is generated during acute inflammation, can act on sensory neurons to induce hyperalgesic priming by activating neuronal PKCɛ.

Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCε second messenger pathways

&NA; Toward the goal of defining new pharmacological targets for the treatment of chronic pain conditions, in previous studies we established a model, termed ‘hyperalgesic priming,’ in which an acute inflammatory stimulus causes a long‐lasting latent susceptibility to hyperalgesia induced by subsequent exposures to the inflammatory mediator, prostaglandin E2 (PGE2). Those investigations suggested the hypothesis that priming induces a novel linkage between the PGE2‐activated second messenger cascade and the epsilon isoform of protein kinase C (PKCϵ). In the present study, comparison of dose–response relations for hyperalgesia produced by PGE2, forskolin, 8‐Br‐cAMP, or the protein kinase A (PKA) catalytic subunit, in primed versus normal animals, demonstrated that priming‐induced enhancement of the PGE2‐activated second messenger cascade occurs downstream to adenylate cyclase and upstream to PKA. Therefore, PGE2‐induced hyperalgesia in the primed animal is enhanced by the recruitment of a novel cAMP/PKCϵ signaling pathway in addition to the usual cAMP/PKA pathway. These observations suggest that pharmacological disruption of the novel interaction between cAMP and PKCϵ might provide a route toward the development of highly specific methods to reverse cellular processes that underlie chronic pain states.

Transient attenuation of protein kinase Cϵ can terminate a chronic hyperalgesic state in the rat

Abstract Recently we demonstrated that a single 3-day episode of carrageenan-induced acute cutaneous inflammation can create a chronic state of increased susceptibility to inflammatory hyperalgesia. In this latent “primed” state, although there is no ongoing hyperalgesia, the hyperalgesic response to subsequent challenges with inflammatory agent (prostaglandin E 2 ; PGE 2 ) is greatly enhanced. Furthermore, the PGE 2 -induced hyperalgesia in primed skin was found to require activity of the ϵ isozyme of protein kinase C (PKCϵ), a second messenger that is not required for PGE 2 -induced hyperalgesia in control animals. In the present study we tested the hypothesis that activity of PKCϵ not only plays a critical role in the expression of primed PGE 2 -induced hyperalgesia, but also in the development and maintenance of the primed state itself. Antisense oligodeoxynucleotide was employed to produce a decrease in PKCϵ in the nerve, verified by Western blot analysis. PKCϵ was found to be essential both for the development of carrageenan-induced hyperalgesic priming, as well as for the maintenance of the primed state. Furthermore, hyperalgesic priming could be induced by an agonist of PKCϵ (pseudo-receptor octapeptide for activated PKCϵ) at a dose that itself causes no hyperalgesia. The finding that transient inhibition of PKCϵ can not only prevent the development of priming, but can also terminate a fully developed state of priming suggests the possibility that selective targeting PKCϵ might be an effective new strategy in the treatment of chronic inflammatory pain.

Integrin signaling in inflammatory and neuropathic pain in the rat

Many painful conditions are associated with alterations in the extracellular matrix (ECM) of affected tissues. While several integrins, the receptors for ECM proteins, are present on sensory neurons that mediate pain, the possible role of these cell adhesion molecules in inflammatory or neuropathic pain has not been explored. We found that the intradermal injection of peptide fragments of domains of laminin and fibronectin important for adhesive signaling selectively inhibited the hyperalgesia caused by prostaglandin E2 (PGE2) and epinephrine (EPI), respectively. The block of EPI hyperalgesia was mimicked by other peptides containing the RGD integrin‐binding sequence. Monoclonal antibodies (mAbs) against the α1 or α3 integrin subunits, which participate in laminin binding, selectively blocked PGE2 hyperalgesia, while a mAb against the α5 subunit, which participates in fibronectin binding, blocked only EPI‐induced hyperalgesia. A mAb against the β1 integrin subunit, common to receptors for both laminin and fibronectin, inhibited hyperalgesia caused by both agents, as did the knockdown of β1 integrin expression by intrathecal injection of antisense oligodeoxynucleotides. The laminin peptide, but not the fibronectin peptides, also reversibly abolished the longer lasting inflammatory hyperalgesia induced by carrageenan. Finally, the neuropathic hyperalgesia caused by systemic administration of the cancer chemotherapy agent taxol was reversibly inhibited by antisense knockdown of β1 integrin. These results strongly implicate specific integrins in the maintenance of inflammatory and neuropathic hyperalgesia.

Activation of presynaptic NMDA receptors coupled to NaV1.8-resistant sodium channel C-fibers causes retrograde mechanical nociceptor sensitization

The present study investigated whether activation of presynaptic N-methyl-d-aspartate (NMDA) receptors in the spinal cord produces a retrograde nociceptor sensitization (hypernociception) to mechanical nonnoxious stimulus. By using an electronic version of the von Frey hair test (pressure meter), s.c. intraplantar administration of prostaglandin E2 (PGE2) (50–400 ng per paw) evoked a dose-related ipsilateral paw hypernociception. In contrast, intrathecal (i.t.) administration of NMDA (5–80 ng) and PGE2 (15–150 ng) evoked dose-related bilateral paw hypernociception. The s.c. intraplantar administration of dipyrone (80–320 μg per paw) or morphine (3 and 9 μg per paw), usually used to antagonize peripheral PGE2 (100 ng per paw), induced hypernociception and also antagonized the ipsilateral (without affecting the contralateral) paw hypernociception induced by i.t. injections of NMDA (40 ng) or PGE2 (50 ng). These doses of drugs did not modify the basal mechanical sensitivity of control paws. This result shows that intraspinal NMDA or PGE2 produces sensitization of the primary sensory neuron in response to mechanical stimulation. In a second series of experiments it was shown that the i.t. treatment with NaV1.8 (SNS/PN3) sodium channel antisense oligodeoxynucleotides, but not mismatch oligodeoxynucleotides, decreased the mRNA expression of sodium tetrodotoxin-resistant channels on the dorsal root ganglia and abolished the mechanical hypernociception induced by i.t. administration of NMDA. Thus, our results support the suggestion that glutamate release in the spinal cord during inflammation causes retrograde hypernociception of nociceptors associated with sodium tetrodotoxin-resistant channels in primary nociceptive sensory neurons.

Hyperalgesic priming in the rat demonstrates marked sexual dimorphism

In male rats, carrageenan (CAR)‐induced inflammation or exposure to a selective protein kinase C epsilon (PKC&egr;) agonist (&psgr;&egr;RACK) produces prolongation of the hyperalgesia induced by a subsequent exposure to an inflammatory mediator, a phenomenon referred to as hyperalgesic priming. Since many chronic inflammatory conditions are sexually dimorphic, we tested the hypothesis that hyperalgesic priming is sexually dimorphic. Prior injection of CAR or &psgr;&egr;RACK produced a prolongation of the hyperalgesia induced by a subsequent injection of prostaglandin E2, from less than 3 h to greater than 24 h, but only in male rats. In ovariectomized female rats priming with CAR and &psgr;&egr;RACK produced hyperalgesic priming effects similar to that observed in the male rat, and this effect was reversed by estrogen replacement. While gonadectomy in males had no effect on CAR and &psgr;&egr;RACK induced hyperalgesic priming, female phenotype was observed following implantation of estrogen in males. Thus, mechanisms mediating the development of hyperalgesic priming produced by inflammation are suppressed by estrogen. This regulation of priming by estrogen appears to occur at or downstream of the activation of PKC&egr;.

Inhibition of tonic spinal glutamatergic activity induces antinociception in the rat

Inhibition of tonic activity in spino‐supraspinal projection neurons induces heterosegmental antinociception that is mediated by opioid receptors in nucleus accumbens. To investigate the origin of this tonic activity, we evaluated the ability of inhibiting neurotransmission in the spinal cord to produce heterosegmental antinociception in the trigeminal nociceptive jaw‐opening reflex (JOR) in the rat. Spinal intrathecal administration of calcium channel blockers attenuated the JOR, suggesting that the tonic spinal activity depends on synaptic input. To identify the excitatory neurotransmitter receptors involved, selective antagonists for AMPA/kainate, mGluR1, NMDA or NK1 receptors were administered intrathecally to the spinal cord. The AMPA/kainate and mGluR1 receptor antagonists, but not the NMDA or NK1 receptor antagonists, induced antinociception, which was antagonized by intra‐accumbens administration of the selective µ‐opioid receptor antagonist CTOP. Thus, inhibition of tonic spinal glutamatergic activity resulted in supraspinally mediated antinociception. As this antinociception occurred in the absence of interventions that would produce a facilitated nociceptive state, this tonic glutamatergic activity is important in setting nociceptive threshold.

Corrigendum to “Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCε second messenger pathways” [Pain 113 (2005) 185–190]

Fig. 3. Linkage between cAMP and PKC3 in hyperalgesic priming. (A) In primed rats, co-administration of PKC3 inhibitor (PKC3I) significantly decreased hyperalgesia induced by 8-Br-cAMP (10 ng), but in control unprimed rats, had no significant effect on hyperalgesia induced by the same dose of 8-Br-cAMP. (B) In control unprimed rats, A-kinase anchoring protein inhibitor (AKAPI) prevented the hyperalgesia induced by 8-Br-cAMP (10 ng), but only partially decreased 8-Br-cAMP-induced-hyperalgesia in primed rats. Only the combination of AKAPI (1 mg) and PKC3I (1 mg) was able to prevent the hyperalgesia induced by 8-Br-cAMP in primed rats. The symbols *, #, and ** denote values significantly different from 0.01 mg, from saline; and from *, respectively (P!0.05, ANOVA/Tukey’s test, nZ4–10). There was no significant difference between groups marked by the symbols * or ** or #.

Local administration of mangiferin prevents experimental inflammatory mechanical hyperalgesia through CINC-1/epinephrine/PKA pathway and TNF-α inhibition

Abstract Steroidal and non‐steroidal anti‐inflammatory drugs (NSAIDs) are widely used to control inflammatory pain, but there is a risk of gastrointestinal bleeding and increased heart failure risk. The search for new drugs remains ongoing, and natural products are a source for potential new compounds. Mangiferin, a natural xanthone C‐glucoside, has demonstrated biological activity, including anti‐inflammatory and analgesic properties, but its mechanisms are poorly understood. In this study, we investigated the mechanisms underlying the anti‐inflammatory and analgesic effects of local administration of mangiferin. We employed an electronic von Frey apparatus to evaluate mechanical hyperalgesia induced by carrageenan in rats. Mangiferin (150–1200 &mgr;g/paw), administered locally into the hindpaw, prevented hyperalgesia in a dose‐dependent – 150 &mgr;g (− 9%), 300 &mgr;g (− 27%, P < 0.01), 600 &mgr;g (− 77%, P < 0.001) and 1000 &mgr;g (− 93%, P < 0.001) – and local manner. Mangiferin showed decreased levels of TNF‐&agr; (P < 0.001) and CINC‐1 (P < 0.001), but not IL‐1&bgr;; it also prevented neutrophil migration (P < 0.01), but not the increased COX‐2 expression in peripheral tissue challenged with carrageenan. To further explore the mechanisms of mangiferin actions, rats were injected with modulators of inflammation and nociception; mangiferin prevented hyperalgesia induced by IL‐1&bgr; (P < 0.01), CINC‐1 (P < 0.01), epinephrine (P < 0.01), 8‐Br‐cAMP (P < 0.01) or capsaicin (P < 0.01), but not that induced by PGE2 or &agr;,&bgr;‐MeATP. Our study shows that mangiferin has anti‐inflammatory and analgesic properties when locally administrated. The control of the inflammatory response and mechanical hyperalgesia by mangiferin depends on the inhibition of TNF‐&agr; production/release and the CINC1/epinephrine/PKA pathway, supporting its marked inhibition of inflammatory mechanical hyperalgesia.