Formalin-induced Secondary Allodynia Research Articles

Synergistic Interaction of a Gabapentin- Mangiferin Combination in Formalin-Induced Secondary Mechanical Allodynia and Hyperalgesia in Rats Is Mediated by Activation of NO-Cyclic GMP-ATP-Sensitive K+ Channel Pathway.

Preclinical Research Gabapentin is an anticonvulsant used to treat neuropathic pain. Mangiferin is an antioxidant that has antinociceptive and antiallodynic effects in inflammatory and neuropathic pain models. The purpose of this study was to determine the interaction between mangiferin and gabapentin in the development and maintenance of formalin-induced secondary allodynia and hyperalgesia in rats. Gabapentin, mangiferin, or their fixed-dose ratio combination were administrated peripherally. Isobolographic analyses was used to define the nature of the interaction of antiallodynic and/or antihyperalgesic effects of the two compounds. Theoretical ED50 values for the combination were 74.31 µg/paw and 95.20 µg/paw for pre- and post-treatment, respectively. These values were higher than the experimental ED50 values, 29.45 µg/paw and 37.73 µg/paw respectively, indicating a synergistic interaction in formalin-induced secondary allodynia and hyperalgesia. The antiallodynic and antihyperalgesic effect induced by the gabapentin/mangiferin combination was blocked by administration of L-NAME, the soluble guanylyl cyclase inhibitor, ODQ and glibenclamide. These data suggest that the gabapentin- mangiferin combination produces a synergistic interaction at the peripheral level. Moreover, the antiallodynic and hyperalgesic effect induced by the combination is mediated via the activation of an NO-cyclic GMP-ATP-sensitive K+ channel pathway. Drug Dev Res 78 : 390-402, 2017. © 2017 Wiley Periodicals, Inc.

Role of spinal 5-HT2 receptors subtypes in formalin-induced long-lasting hypersensitivity.

The purpose of this study was to determine the role of spinal 5-HT2A, 5-HT2B and 5-HT2C receptors in the development and maintenance of formalin-induced long-lasting secondary allodynia and hyperalgesia in rats, as well as their expression in the dorsal root ganglia (DRG) during this process. 0.5-1% formalin was used to produce long-lasting secondary allodynia and hyperalgesia in rats. Western blot was used to determine 5-HT2 receptors expression in DRG. Formalin (0.5-1%) injection produced long-lasting (1-12 days) secondary allodynia and hyperalgesia in both ipsilateral and contralateral hind paws. Intrathecal pre-treatment or post-treatment with the 5-HT2 receptor agonist, DOI (1-10nmol), increased 0.5% formalin-induced secondary allodynia and hyperalgesia in both paws. In contrast, intrathecal pre-treatment with the selective 5-HT2A (ketanserin 1-100nmol), 5-HT2B (RS 127445 1-100nmol) or 5-HT2C (RS 102221 1-100nmol) receptor antagonists prevented and reversed, respectively, 1% formalin-induced secondary allodynia and hyperalgesia in both paws. Likewise, the pronociceptive effect of DOI (10nmol) was blocked by ketanserin, RS 127445 or RS 102221 (0.01nmol). 5-HT2A/2B/2C receptors were expressed in DRG of naïve rats. Formalin injection (1%) increased bilaterally 5-HT2A/2B receptors expression in DRG. In contrast, formalin injection decreased 5-HT2C receptors expression bilaterally in DRG. Data suggest that spinal 5-HT2A/2B/2C receptors have pronociceptive effects and participate in the development and maintenance of formalin-induced long-lasting hypersensitivity. These receptors are expressed in DRG and their expression is modulated by formalin.

Role of TRPV1 and ASIC3 in formalin-induced secondary allodynia and hyperalgesia

BackgroundIn the present study we determined the role of transient receptor potential V1 channel (TRPV1) and acid-sensing ion channel 3 (ASIC3) in chronic nociception. Methods1% formalin was used to produce long-lasting secondary allodynia and hyperalgesia in rats. Western blot was used to determine TRPV1 and ASIC3 expression in dorsal root ganglia. ResultsPeripheral ipsilateral, but not contralateral, pre-treatment (−10min) with the TRPV1 receptor antagonists capsazepine (0.03–0.3μM/paw) and A-784168 (0.01–1μM/paw) prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in the ipsilateral and contralateral paws. Likewise, peripheral ipsilateral, but not contralateral, pre-treatment with the non-selective and selective ASIC3 blocker benzamil (0.1–10μM/paw) and APETx2 (0.02–2μM/paw), respectively, prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Peripheral ipsilateral post-treatment (day 6 after formalin injection) with capsazepine (0.03–0.3μM/paw) and A-784168 (0.01–1μM/paw) reversed 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. In addition, peripheral ipsilateral post-treatment with benzamil (0.1–10μM/paw) and APETx2 (0.02–2μM/paw), respectively, reversed 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. TRPV1 and ASIC3 proteins were expressed in dorsal root ganglion in normal conditions, and 1% formalin injection increased expression of both proteins in this location at 1 and 6 days compared to naive rats. ConclusionsData suggest that TRPV1 and ASIC3 participate in the development and maintenance of long-lasting secondary allodynia and hyperalgesia induced by formalin in rats. The use of TRPV1 and ASIC3 antagonists by peripheral administration could prove useful to treat chronic pain.

Role of glutamate receptors in the dorsal reticular nucleus in formalin‐induced secondary allodynia

The role of glutamate receptors present in the medullary dorsal reticular nucleus (DRt) in the formalin test and formalin-induced secondary nociception was studied in rats. Secondary mechanical allodynia was assessed with von Frey filaments applied to the rat's hindpaw, and secondary thermal hyperalgesia was evaluated with the tail-immersion test. The selective glutamate receptor antagonists MK801 (N-methyl-D-aspartate receptor antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (AMPA/KA receptor antagonist) and A841720 (metabotropic glutamate 1 receptor antagonist) were injected into the DRt before or 6 days after formalin injection in the rat. In the formalin test, the three antagonists significantly reduced the number of flinches in both phases of the test. DRt microinjection of MK801 or A841720, but not of CNQX, reduced both secondary nociceptive behaviors. Moreover, pre-treatment with the three antagonists injected into the DRt prevented the development of secondary mechanical allodynia and secondary thermal hyperalgesia. Similarly, in these rats, the number of c-Fos-like immunoreactive neurons were markedly reduced in both the superficial and deep lamina of the dorsal horn. Our findings support the role of DRt as a pain facilitator in acute and chronic pain states, and suggest a key role of glutamate receptors during the development and maintenance of formalin-induced secondary allodynia.

Evidence for the participation of peripheral 5-HT2A, 5-HT2B, and 5-HT2C receptors in formalin-induced secondary mechanical allodynia and hyperalgesia

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 10 μg/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 (10 nmol/paw), 5-HT (100 nmol/paw) and fluoxetine (1 nmol/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.

Secondary mechanical allodynia and hyperalgesia depend on descending facilitation mediated by spinal 5-HT4, 5-HT6 and 5-HT7 receptors

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.

Role of peripheral and spinal 5-HT3 receptors in development and maintenance of formalin-induced long-term secondary allodynia and hyperalgesia

The role of peripheral and spinal 5-HT3 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 in both paws. In experiments where the test drug was anticipated to augment or antagonize the response, 0.5 or 1% formalin, respectively, was used for injection. Peripheral ipsilateral, but not contralateral, pre-treatment (−10min) with serotonin (5-HT, 10–100nmol/paw) and the selective 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide (m-CPBG, 10–300nmol/paw) increased 0.5% formalin-induced secondary allodynia and hyperalgesia in both paws. Moreover, spinal pre-treatment with m-CPBG (10–300nmol/rat) increased 0.5% formalin-induced secondary hyperalgesia but not allodynia in both paws. Accordingly, peripheral ipsilateral (30–300nmol/paw), but not contralateral (300nmol/paw), and spinal (10–100nmol) pre-treatment with the selective 5-HT3 receptor antagonist ondansetron prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. The peripheral pronociceptive effects of 5-HT (100nmol/paw) and m-CPBG (300nmol/paw) as well as the spinal effect of m-CPBG (300nmol/rat) were completely prevented by the peripheral (10nmol/paw) and spinal (1nmol/rat) injection, respectively, of ondansetron. At these doses, ondansetron did not modify per se formalin-induced nociceptive behaviors. Spinal (30–300nmol/rat), but not peripheral (300nmol/paw), post-treatment (on day 6) with ondansetron reversed established formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Results suggest that a barrage of afferent input induced by 5-HT at peripheral 5-HT3 receptors participates in the development of formalin-induced long-term secondary allodynia and hyperalgesia in the rat. In addition, our data suggest that spinal 5-HT3 receptors play an important role during development and maintenance of these evoked long-term behaviors.

Chromaffin cell transplant in spinal cord reduces secondary allodynia induced by formalin in the rat. Role of opioid receptors and α2-adrenoceptors

In the present study, the effect of chromaffin cell transplant in the spinal cord was evaluated on formalin-induced mechanical secondary allodynia in the rat. Chromaffin cells were transplanted into the lumbar subarachnoid space before or after formalin injection. Subcutaneous formalin injection (50μl, 1%) produced long-lasting secondary allodynia in the ipsilateral and contralateral hind paws. Once secondary allodynia was established, treatment with chromaffin cells produced a significant reduction in the nociceptive behavior in both hind paws. The antiallodynic effect was time-dependent since it was observed 15days after chromaffin cell transplants but not before. On the other hand, pre-treatment with chromaffin cells prevented the expression of secondary allodynia in both hind paws in the rat. Antiallodynic effect of chromaffin cells was reverted with the non-selective opioid receptor antagonist naltrexone and the non-selective α2-adrenoceptor antagonist rauwolscine. Clusters of viable chromaffin cells labeled with anti-tyrosine hydroxylase antibodies were observed in the retrieved transplants 15days after transplant. These results establish the analgesic efficacy of intrathecal chromaffin cells on formalin-induced secondary allodynia. Our data suggest that chromaffin cells release neuroactive substances including opioid peptides and adrenergic amines that reduce secondary allodynia in rats through activation of their receptors.

Formalin-induced long-term secondary allodynia and hyperalgesia are maintained by descending facilitation

This work analyzes the role of cholecystokinin (CCK) receptors, dynorphin A1–17 and descending facilitation originated in the rostral ventromedial medulla (RVM) on secondary allodynia and hyperalgesia in formalin-injected rats. Formalin injection (50μL, 1%, s.c.) produced acute nociception (lasting 1h) and long-term secondary allodynia and hyperalgesia in ipsilateral and contralateral hind paws (lasting 1–12days). Once established, intra-RVM administration of lidocaine at day 6, but not at 2, reversed secondary allodynia and hyperalgesia in rats. The injection of YM022 (CCK2 receptor antagonist), but not lorglumide (CCK1 receptor antagonist), into the RVM or spinal cord reversed both nociceptive behaviors. Pre-treatment with lidocaine, lorglumide or YM022 did not prevent the development of secondary allodynia or hyperalgesia regardless of the administration route. Formalin injection increased dynorphin content in the dorsal, but not the ventral, spinal cord sections at day 6. Moreover, intrathecal administration of dynorphin antiserum reversed, but was unable to prevent, secondary allodynia and hyperalgesia in both hind paws. These results suggest that formalin-induced secondary allodynia and hyperalgesia are maintained by activation of descending facilitatory mechanisms which are dependent on CCK2 receptors located in the RVM and spinal cord. In addition, data suggest that spinal dynorphin A1–17 and CCK play an important role in formalin-induced secondary allodynia and hyperalgesia.

Role of peripheral 5-HT4, 5-HT6, and 5-HT7 receptors in development and maintenance of secondary mechanical allodynia and hyperalgesia

The role of 5-hydroxytryptamine (5-HT)4, 5-HT6, and 5-HT7 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. Pretreatment (−10min) with cromoglycate (195–1950nmol/paw) partially inhibited acute nociceptive behaviors and completely prevented secondary allodynia and hyperalgesia on day 6 after injection. Ipsilateral peripheral pretreatment with the selective 5-HT4 (ML-10302, 1–100nmol/paw), 5-HT6 (EMD-386088, 0.001–0.01nmol/paw), and 5-HT7 (LP-12, 0.01–100nmol/paw) receptor agonists significantly increased secondary allodynia and hyperalgesia in both paws. In contrast, ipsilateral peripheral pretreatment with the selective 5-HT4 (GR-125487, 1–100nmol/paw), 5-HT6 (SB-258585, 0.00001–0.001nmol/paw), and 5-HT7 (SB-269970, 0.1–10nmol/paw) receptor antagonists significantly prevented formalin-induced secondary allodynia and hyperalgesia in both paws. The pronociceptive effect of ML-10302 (100nmol/paw), EMD-386088 (0.01nmol/paw), and LP-12 (100nmol/paw) were completely prevented by GR-125487 (5-HT4 antagonist, 1nmol/paw), SB-258585 (5-HT6 antagonist, 0.00001nmol/paw), and SB-269970 (5-HT7, antagonist, 0.01nmol/paw), respectively. Ipsilateral peripheral posttreatment with cromoglycate or GR-125487 (1–100nmol/paw), SB-258585 (0.001–0.1nmol/paw), and SB-269970 (0.1–10nmol/paw) reversed formalin-induced secondary allodynia and hyperalgesia in both paws. Results suggest that a barrage of afferent input induced by 5-HT at peripheral 5-HT4, 5-HT6, and 5-HT7 receptors participate in the development and maintenance of formalin-induced long-term secondary allodynia and hyperalgesia in the rat.5-hydroxytryptamine (5-HT) released in peripheral tissues after formalin injection sensitized primary afferent neurons via 5-HT4, 5-HT6, and 5-HT7 receptors, leading to development and maintenance of secondary allodynia and hyperalgesia.

Role of opioid receptors in the reduction of formalin-induced secondary allodynia and hyperalgesia in rats

This study assesses the effects of peripheral or intrathecal pre-treatment or post-treatment with µ, δ, κ and nociceptin/orphanin FQ (NOP) opioid receptor agonists (morphine, U-50488 [trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride], DADLE [D-Ala2-Leu5-enkephalin] and nociceptin, respectively) on formalin-induced secondary mechanical allodynia and hyperalgesia in rats. 1% Formalin injection produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term tactile secondary allodynia and hyperalgesia. Neither peripheral (into the formalin-injected paw) nor intrathecal morphine post-treatment reversed formalin-induced secondary allodynia and hyperalgesia. In contrast, morphine pre-treatment prevented the development of these pain behaviors. Intrathecal and peripheral post- but not pre-treatment with U-50488 or DADLE significantly reduced secondary allodynia and hyperalgesia. Interestingly, nociceptin reduced both pain behaviors regardless of the administration site or treatment time. Local antinociceptive effects of morphine, DADLE, U-50488 or nociceptin were blocked by naltrexone, naltrindole, 5-guanidinonaltrindole and [Nphe 1]nociceptin(1–13)NH 2, respectively. These results suggest that the long-term nociceptive behaviors induced by formalin are differentially modulated by selective opioid receptor agonists. In addition, data suggest that peripheral and spinal δ and κ opioid receptors are important when nociceptive behaviors are established. In contrast, µ opioid receptors are more important at the beginning of the injury when the sensory system has not changed. NOP receptors participate diminishing both the development and maintenance of nociceptive behaviors. Results suggest that a barrage of afferent input induced by formalin injection initiates a long-term differential change in peripheral and spinal processing that affect the efficacy of opioid receptor agonists.

P.2.d.020 Antidepressant-like effect of guanosine in the forced swimming test and in the tail suspension test

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 10 μg/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 (10 nmol/paw), 5-HT (100 nmol/paw) and fluoxetine (1 nmol/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.