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Vladimir A Martínez-Rojas,1 Guadalupe García,1 Roxana Noriega-Navarro,1 Crystell G Guzmán-Priego,2 Jorge E Torres-López,2,3 Vinicio Granados-Soto,4 Janet Murbartián1 1Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados (Cinvestav), Unidad Coapa, Ciudad de México, 2Laboratorio Mecanismos del Dolor, Centro de Investigación, División Académica de Ciencias de la Salud, Universidad Juárez Autónoma de Tabasco, 3Hospital Regional de Alta Especialidad “Dr. Juan Graham Casasús”, Villahermosa, Tabasco, 4Neurobiology of Pain Laboratory, Departamento de Farmacobiología, Cinvestav, Unidad Coapa, Ciudad de México, México Background: Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed by a subset of nociceptive neurons that acts as a multimodal receptor. Its activity contributes to modulate nociceptive transmission in acute inflammatory pain. However, the role of this channel in chronic pain has been less studied. The purpose of this study was to investigate the local peripheral and spinal participation of TRPA1 channels in formalin-induced long-lasting hypersensitivity.Materials and methods: Formalin (1%)-induced chronic hypersensitivity was determined by the application of von Frey filaments to ipsilateral and contralateral paws and through pharmacological testing using a selective TRPA1 blocker (A-967079). TRPA1 expression in the dorsal root ganglion (DRG) and spinal cord was analyzed by Western blotting.Results: Formalin (1%) injection produced acute flinching behavior (1 h) as well as secondary allodynia and hyperalgesia (12 days). Local peripheral pretreatment (10 min before) or posttreatment (6 days later) with A-967079 (1–100 µM) partially prevented and reversed, respectively, in a dose-dependent manner, long-lasting secondary mechanical allodynia and hyperalgesia evoked by 1% formalin. Likewise, intrathecal pretreatment or posttreatment with A-967079 partially prevented and reversed, respectively, formalin-induced long-lasting hypersensitivity. A-967079 (100 µM) completely abolished the pro-nociceptive effect of formalin (adjusted to pH 7.4). Finally, formalin injection increased TRPA1 protein expression in the DRG and spinal cord.Conclusion: Results indicate that TRPA1 expressed in the DRG and spinal cord plays a relevant role in formalin-induced long-lasting secondary nociceptive hypersensitivity. Keywords: allodynia, chronic pain, formalin, hyperalgesia, TRPA1

Abstract: This study assessed the possible antinociceptive role of peripheral 5-HT1 receptor subtypes in the rat formalin test. Rats were injected into the dorsum of the hind paw with 50 μl of diluted formalin (1%). Nociceptive behavior was quantified as the number of flinches of the injected paw. Reduction of flinching was considered as antinociception. Ipsilateral, but not contralateral, peripheral administration of the 5-HT1 receptor agonists R(+)-UH-301 (5-HT1A; 0.1–3 μg/paw), CGS-12066A (5-HT1B; 0.01–0.3 μg/paw), GR46611 (5-HT1B/1D; 0.3–10 μg/paw), BRL54443 (5-HT1E/1F; 3–300 μg/paw) or LY344864 (5-HT1F; 3–300 μg/paw) significantly reduced formalin-induced flinching. The corresponding vehicle was devoid of any effect by itself. The local antinociceptive effect of R(+)-UH-301 (0.3 μg/paw) was significantly reduced by WAY-100635 (30–100 μg/paw; a 5-HT1A receptor antagonist). Moreover, the antagonists GR55562 (30–100 μg/paw; 5-HT1B/D) or SB224289 (30–100 μg/paw; 5-HT1B) dose-dependently reduced the antinociceptive effect of CGS-12066A (0.3 μg/paw) whereas GR55562 (30–100 μg/paw) or BRL15572 (30–100 μg/paw, 5-HT1D) reduced the antinociceptive effect of GR46611 (0.3 μg/paw). Interestingly, the effects of BRL54443 and LY344864 (300 μg/paw each) were partially reduced by methiothepin, but not by the highest doses of WAY-100635, SB224289 or BRL15572. The above antagonists did not produce any effect by themselves. These results suggest that peripheral activation of the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F and, probably, 5-HT1E receptor subtypes leads to antinociception in the rat formalin test. Thus, the use of selective 5-HT1 receptor agonists could be a therapeutic strategy to reduce inflammatory pain. [Copyright &y& Elsevier]

Abstract: The role of 5-HT2A/2B/2C receptors in formalin-induced secondary allodynia and hyperalgesia in rats was assessed. Formalin produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term secondary mechanical allodynia and hyperalgesia. Pre-treatment for five consecutive days with compound 48/80 (1, 3, 10, 10, and 10μg/paw) prevented formalin-induced secondary allodynia and hyperalgesia. Ipsilateral, but not contralateral, peripheral pre-treatment (nmol/paw) with the 5-HT2 receptor agonist DOI (3-30), 5-HT (10-100) or fluoxetine (0.3-3) significantly increased 0.5% formalin-induced secondary allodynia and hyperalgesia in both paws. The pronociceptive effect of DOI (10nmol/paw), 5-HT (100nmol/paw) and fluoxetine (1nmol/paw) was blocked by selective 5-HT2A (ketanserin), 5-HT2B (RS-127445), and 5-HT2C (RS-102221) receptor antagonists. Furthermore, ipsilateral pre-treatment (nmol/paw) with ketanserin (1, 10, and 100), RS-127445 (0.01, 0.1 and 1) or RS-102221 (1, 10 and 100) prevented while post-treatment reversed 1% formalin-induced secondary allodynia and hyperalgesia in both paws. In marked contrast, contralateral injection of the greatest tested dose of 5-HT2A/2B/2C receptor antagonists did not modify long-lasting secondary allodynia and hyperalgesia. These results suggest that 5-HT released from mast cells after formalin injection sensitizes primary afferent neurons via 5-HT2A/2B/2C receptors leading to the development and maintenance of secondary allodynia and hyperalgesia. [Copyright &y& Elsevier]

Abstract: In the present study we determined the role of spinal 5-hydroxytriptamine (5-HT) and 5-HT4/6/7 receptors in the long-term secondary mechanical allodynia and hyperalgesia induced by formalin in the rat. Formalin produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term secondary mechanical allodynia and hyperalgesia in both paws. In addition, formalin increased the tissue content of 5-HT in the ipsilateral, but not contralateral, dorsal part of the spinal cord compared to control animals. Intrathecal (i.t.) administration of 5,7-dihydroxytriptamine (5,7-DHT), a serotonergic neurotoxin, diminished tissue 5-HT content in the ipsilateral and contralateral dorsal parts of the spinal cord. Accordingly, i.t. 5,7-DHT prevented formalin-induced secondary allodynia and hyperalgesia in both paws. I.t. pre-treatment (−10min) with ML-10302 (5-HT4 agonist), EMD-386088 (5-HT6 agonist) and LP-12 (5-HT7 agonist) significantly increased secondary mechanical allodynia and hyperalgesia in both paws. In contrast, i.t. pre-treatment (−20min) with GR-125487 (5-HT4 antagonist), SB-258585 (5-HT6 antagonist) and SB-269970 (5-HT7 antagonist) significantly prevented formalin-induced long-term effects in both paws. In addition, these antagonists prevented the pro-nociceptive effect of ML-10302, EMD-386088 and LP-12, respectively. The i.t. post-treatment (6days after formalin injection) with GR-125487, SB-258585 and SB-269970 reversed formalin-induced secondary allodynia and hyperalgesia in both paws. These results suggest that spinal 5-HT, released from the serotonergic projections in response to formalin injection, activates pre- or post-synaptic 5-HT4/6/7 receptors at the dorsal root ganglion/spinal cord promoting the development and maintenance of secondary allodynia and hyperalgesia. [Copyright &y& Elsevier]

Abstract: The present study assessed the possible pronociceptive role of peripheral and spinal 5-HT6 receptors in the formalin test. For this, local peripheral administration of selective 5-HT6 receptor antagonists N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)-benzenesulphonamide (SB-399885) (0.01–1 nmol/paw) and 4-iodo-N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]benzene-sulfonamide hydrochloride (SB-258585) (0.001–0.1 nmol/paw) significantly reduced formalin-induced flinching. Local peripheral serotonin (5-HT) (10–100 nmol/paw) or 5-chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole hydrochloride (EMD-386088) (0.01–0.1 nmol/paw; a selective 5-HT6 receptor agonist) augmented 0.5% formalin-induced nociceptive behavior. The local pronociceptive effect of 5-HT (100 nmol/paw) or EMD-386088 (0.1 nmol/paw) was significantly reduced by SB-399885 or SB-258585 (0.1 nmol/paw). In contrast to peripheral administration, intrathecal injection of 5-HT6 receptor antagonists SB-399885 and SB-258585 (0.1–10 nmol/rat) did not modify 1% formalin-induced nociceptive behavior. Spinal 5-HT (50–200 nmol/rat) significantly reduced formalin-induced flinching behavior during phases 1 and 2. Contrariwise, intrathecal EMD-386088 (0.1–10 nmol/rat) dose-dependently increased flinching during phase 2. The spinal pronociceptive effect of EMD-386088 (1 nmol/rat) was reduced by SB-399885 (1 nmol/rat) and SB-258585 (0.1 nmol/rat). Our results suggest that 5-HT6 receptors play a pronociceptive role in peripheral as well as spinal sites in the rat formalin test. Thus, 5-HT6 receptors could be a target to develop analgesic drugs. [Copyright &y& Elsevier]

Abstract: mRNA and protein presence of Na+/H+ exchanger (NHE) 1 (NHE1) and 5 (NHE5) in dorsal root ganglion (DRG) and dorsal spinal cord as well as its possible role in three inflammatory nociception tests were determined. Local peripheral ipsilateral, but not contralateral, administration of NHE inhibitors 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.3–30 μM/paw), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 0.3–30 μM/paw) and amiloride (0.1–10 μM/paw) significantly increased flinching but not licking behavior in the capsaicin and 5-HT tests. Moreover, DMA and EIPA (0.03–30 μM/paw) as well as amiloride (0.1–1 μM/paw) augmented, in a dose-dependent manner, 0.5% formalin-induced flinching behavior during phase II but not during phase I. Reverse transcription–polymerase chain reaction showed the expression of NHE1 and NHE5 in DRG and dorsal spinal cord. Western blot analysis confirmed the presence of NHE1 in DRG and spinal cord. Moreover, NHE5 was expressed in dorsal spinal cord, but not in DRG where a 45 kDa truncated isoform of NHE5 was identified. Collectively, these data suggest that NHE1, but not NHE5, plays an important role reducing inflammatory pain in rats. [Copyright &y& Elsevier]