Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents.

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

Hypothalamic A11 Nuclei Regulate the Circadian Rhythm of Spinal Mechanonociception through Dopamine Receptors and Clock Gene Expression.

Several types of sensory perception have circadian rhythms. The spinal cord can be considered a center for controlling circadian rhythms by changing clock gene expression. However, to date, it is not known if mechanonociception itself has a circadian rhythm. The hypothalamic A11 area represents the primary source of dopamine (DA) in the spinal cord and has been found to be involved in clock gene expression and circadian rhythmicity. Here, we investigate if the paw withdrawal threshold (PWT) has a circadian rhythm, as well as the role of the dopaminergic A11 nucleus, DA, and DA receptors (DR) in the PWT circadian rhythm and if they modify clock gene expression in the lumbar spinal cord. Naïve rats showed a circadian rhythm of the PWT of almost 24 h, beginning during the night-day interphase and peaking at 14.63 h. Similarly, DA and DOPAC's spinal contents increased at dusk and reached their maximum contents at noon. The injection of 6-hydroxydopamine (6-OHDA) into the A11 nucleus completely abolished the circadian rhythm of the PWT, reduced DA tissue content in the lumbar spinal cord, and induced tactile allodynia. Likewise, the repeated intrathecal administration of D1-like and D2-like DA receptor antagonists blunted the circadian rhythm of PWT. 6-OHDA reduced the expression of Clock and Per1 and increased Per2 gene expression during the day. In contrast, 6-OHDA diminished Clock, Bmal, Per1, Per2, Per3, Cry1, and Cry2 at night. The repeated intrathecal administration of the D1-like antagonist (SCH-23390) reduced clock genes throughout the day (Clock and Per2) and throughout the night (Clock, Per2 and Cry1), whereas it increased Bmal and Per1 throughout the day. In contrast, the intrathecal injection of the D2 receptor antagonists (L-741,626) increased the clock genes Bmal, Per2, and Per3 and decreased Per1 throughout the day. This study provides evidence that the circadian rhythm of the PWT results from the descending dopaminergic modulation of spinal clock genes induced by the differential activation of spinal DR.

The role of spinal cord extrasynaptic α5 GABAA receptors in chronic pain.

Chronic pain is an incapacitating condition that affects a large population worldwide. Until now, there is no drug treatment to relieve it. The impairment of GABAergic inhibition mediated by GABAA receptors (GABAA R) is considered a relevant factor in mediating chronic pain. Even though both synaptic and extrasynaptic GABAA inhibition are present in neurons that process nociceptive information, the latter is not considered relevant as a target for the development of pain treatments. In particular, the extrasynaptic α5 GABAA Rs are expressed in laminae I-II of the spinal cord neurons, sensory neurons, and motoneurons. In this review, we discuss evidence showing that blockade of the extrasynaptic α5 GABAA Rs reduces mechanical allodynia in various models of chronic pain and restores the associated loss of rate-dependent depression of the Hoffmann reflex. Furthermore, in healthy animals, extrasynaptic α5 GABAA R blockade induces both allodynia and hyperalgesia. These results indicate that this receptor may have an antinociceptive and pronociceptive role in healthy and chronic pain-affected animals, respectively. We propose a hypothesis to explain the relevant role of the extrasynaptic α5 GABAA Rs in the processing of nociceptive information. The data discussed here strongly suggest that this receptor could be a valid pharmacological target to treat chronic pain states.

Activation of the integrated stress response in nociceptors drives methylglyoxal-induced pain.

Methylglyoxal (MGO) is a reactive glycolytic metabolite associated with painful diabetic neuropathy at plasma concentrations between 500 nM and 5 µM. The mechanisms through which MGO causes neuropathic pain at these pathological concentrations are not known. Because MGO has been linked to diabetic neuropathic pain, which is prevalent and poorly treated, insight into this unsolved biomedical problem could lead to much needed therapeutics. Our experiments provide compelling evidence that ∼1-µM concentrations of MGO activate the integrated stress response (ISR) in IB4-positive nociceptors in the dorsal root ganglion (DRG) of mice in vivo and in vitro. Blocking the integrated stress response with a specific inhibitor (ISRIB) strongly attenuates and reverses MGO-evoked pain. Moreover, ISRIB reduces neuropathic pain induced by diabetes in both mice and rats. Our work elucidates the mechanism of action of MGO in the production of pain at pathophysiologically relevant concentrations and suggests a new pharmacological avenue for the treatment of diabetic and other types of MGO-driven neuropathic pain.

α5GABAA receptors play a pronociceptive role and avoid the rate-dependent depression of the Hoffmann reflex in diabetic neuropathic pain and reduce primary afferent excitability.

Diabetic neuropathy is an incapacitating complication in diabetic patients. The cellular and molecular mechanisms involved in this pathology are poorly understood. Previous studies have suggested that the loss of spinal GABAergic inhibition participate in painful diabetic neuropathy. However, the role of extrasynaptic α5 subunit-containing GABAA (α5GABAA) receptors in this process is not known. The purpose of this study was to investigate the role of α5GABAA receptors in diabetes-induced tactile allodynia, loss of rate-dependent depression (RDD) of the Hoffmann reflex (HR), and modulation of primary afferent excitability. Intraperitoneal administration of streptozotocin induced tactile allodynia. Intrathecal injection of α5GABAA receptor inverse agonist, L-655,708, produced tactile allodynia in naive rats, whereas it reduced allodynia in diabetic rats. In healthy rats, electrical stimulation of the tibial nerve at 5 Hz induced RDD of the HR, although intrathecal treatment with L-655,708 (15 nmol) abolished RDD of the HR. Streptozotocin induced the loss of RDD of the HR, while intrathecal L-655,708 (15 nmol) restored RDD of the HR. L-655,708 (15 nmol) increased tonic excitability of the primary afferents without affecting the phasic excitability produced by the primary afferent depolarization. α5GABAA receptors were immunolocalized in superficial laminae of the dorsal horn and L4 to L6 dorsal root ganglion. Streptozotocin increased mean fluorescence intensity and percentage of neurons expressing α5GABAA receptors in dorsal horn and L4 to L6 dorsal root ganglia in 10-week diabetic rats. Our results suggest that spinal α5GABAA receptors modulate the HR, play an antinociceptive and pronociceptive role in healthy and diabetic rats, respectively, and are tonically active in primary afferents.

Pyritinol reduces nociception and oxidative stress in diabetic rats.

The purpose of this study was to assess the antinociceptive and antiallodynic effect of pyritinol as well as its possible mechanism of action in diabetic rats. Streptozotocin (50 mg/kg) injection caused hyperglycemia within 1 week. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats. Oral acute administration of pyritinol (50-200 mg/kg) dose-dependently reduced flinching behavior in diabetic rats. Moreover, prolonged administration of pyritinol (12.5-50 mg/kg, every 2 days for 2 weeks) reduced formalin-induced nociception. 1H-[1,2,4]-oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, a guanylyl cyclase inhibitor, 2 mg/kg, i.p.), but not naltrexone (a non-selective opioid receptor antagonist, 1 mg/kg, s.c.) or indomethacin (a non-selective cycloxygenase inhibitor, 5 mg/kg, i.p.), blocked the pyritinol-induced antinociception in diabetic rats. Given alone ODQ, naltrexone or indomethacin did not modify formalin-induced nociception in diabetic rats. Oral acute (200 mg/kg) or prolonged (25 mg/kg, every 2 days for 2 weeks) administration of pyritinol significantly reduced streptozotocin-induced changes in free carbonyls, dityrosine, malondialdehyde and advanced oxidative protein products. Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats. On this condition, oral administration of pyritinol (50-200 mg/kg) reduced tactile allodynia in diabetic rats. Results indicate that pyritinol is able to reduce formalin-induced nociception and tactile allodynia in streptozotocin-injected rats. In addition, data suggest that activation of guanylyl cyclase and the scavenger properties of pyritinol, but not improvement in glucose levels, play an important role in these effects.

Benfotiamine relieves inflammatory and neuropathic pain in rats.

Benfotiamine has shown therapeutic efficacy in the treatment of painful diabetic neuropathy in human beings. However, so far there is no evidence about the efficacy of this drug in preclinical models of pain. The purpose of this study was to assess the possible antinociceptive and antiallodynic effect of benfotiamine in inflammatory and neuropathic pain models in the rat. Inflammatory pain was induced by injection of formalin in non-diabetic and diabetic (2 weeks) rats. Reduction of flinching behavior was considered as antinociception. Neuropathic pain was induced by either ligation of left L5/L6 spinal nerves or administration of streptozotocin (50 mg/kg, i.p.) in Wistar rats. Benfotiamine significantly reduced inflammatory (10-300 mg/kg) and neuropathic (75-300 mg/kg) nociception in non-diabetic and diabetic rats. Results indicate that oral administration of benfotiamine is able to reduce tactile allodynia from different origin in the rat and they suggest the use of this drug to reduce inflammatory and neuropathic pain in humans.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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 5-HT5A and 5-HT1B/1D receptors in the antinociception produced by ergotamine and valerenic acid in the rat formalin test.

Sumatriptan, dihydroergotamine and methysergide inhibit 1% formalin-induced nociception by activation of peripheral 5-HT1B/1D receptors. This study set out to investigate the pharmacological profile of the antinociception produced by intrathecal and intraplantar administration of ergotamine (a 5-HT1B/1D and 5-HT5A/5B receptor agonist) and valerenic acid (a partial agonist at 5-HT5A receptors). Intraplantar injection of 1% formalin in the right hind paw resulted in spontaneous flinching behavior of the injected hindpaw of female Wistar rats. Intrathecal ergotamine (15nmol) or valerenic acid (1 nmol) blocked in a dose dependent manner formalin-induced nociception. The antinociception by intrathecal ergotamine (15nmol) or valerenic acid (1nmol) was partly or completely blocked by intrathecal administration of the antagonists: (i) methiothepin (non-selective 5-HT5A/5B; 0.01-0.1nmol); (ii) SB-699551 (selective 5-HT5A; up to 10nmol); (iii) anti-5-HT5A antibody; (iv) SB-224289 (selective 5-HT1B; 0.1-1nmol); or (v) BRL-15572 (selective 5-HT1D; 0.1-1nmol). Likewise, antinociception by intraplantar ergotamine (15nmol) and valerenic acid (10nmol) was: (i) partially blocked by methiothepin (1nmol), SB-699551 (10nmol) or SB-224289 (1nmol); and (ii) abolished by BRL-15572 (1nmol). The above doses of antagonists (which did not affect per se the formalin-induced nociception) were high enough to completely block their respective receptors. Our results suggest that ergotamine and valerenic acid produce antinociception via 5-HT5A and 5-HT1B/1D receptors located at both spinal and peripheral sites. This provides new evidence for understanding the modulation of nociceptive pathways in inflammatory pain.

Effect of K+ channel modulators on the antiallodynic effect of gabapentin.

The effect of K+ channel inhibitors on the antiallodynic activity induced by spinal gabapentin was assessed in rats. Ligation of L5 and L6 spinal nerves made the rats allodynic, whereas that intrathecal administration of gabapentin (25-200 microg) reduced tactile allodynia in a dose-dependent manner. Spinal pretreatment with glibenclamide (12.5-50 microg, ATP-sensitive K+ channel inhibitor), charybdotoxin (0.01-1 ng) or apamin (0.1-3 ng, large-and small-conductance Ca2+-activated K+ channel blockers, respectively), but not margatoxin (0.01-10 ng, voltage-dependent K+ channel inhibitor), significantly prevented gabapentin-induced antiallodynia. Pinacidil (1-30 microg, K+ channel opener) significantly reduced nerve ligation-induced allodynia. Intrathecal glibenclamide (50 microg), charybdotoxin (1 ng) and apamin (3 ng), but not margatoxin (10 ng), significantly reduced pinacidil-induced antiallodynia. K+ channel inhibitors alone did not modify allodynia produced by spinal nerve ligation. Results suggest that gabapentin and pinacidil may activate Ca2+-activated and ATP-sensitive K+ channels in order to produce part of its spinal antiallodynic effect in the Chung model.

Pharmacological evidence for the activation of K(+) channels by diclofenac.

The involvement of K(+) channels in the antinociceptive action of diclofenac was assessed in the formalin test. Local administration of diclofenac produced a dose-dependent antinociceptive effect due to a local action because drug administration in the contralateral paw was ineffective. Pretreatment of the injured paw with glibenclamide and tolbutamide (ATP-sensitive K(+) channel inhibitors), charybdotoxin and apamin (large- and small-conductance Ca(2+)-activated K(+) channel blockers, respectively), 4-aminopyridine or tetraethylammonium (voltage-dependent K(+) channel inhibitors) prevented diclofenac-induced antinociception. Given alone, K(+) channel inhibitors did not modify formalin-induced nociceptive behavior. Pinacidil (an ATP-sensitive K(+) channel opener) also produced antinociception which was blocked by glibenclamide. The peripheral antinociceptive effect of morphine (positive control) was blocked by glibenclamide and 4-aminopyridine but not by charybdotoxin or apamin. The results suggest that the peripheral antinociceptive effect of diclofenac may result from the activation of several types of K(+) channels, which may cause hyperpolarization of peripheral terminals of primary afferents.

Spinal 5-HT5A receptors mediate 5-HT-induced antinociception in several pain models in rats.

The antinociceptive role of spinal 5-HT5A receptors in rat models of pain along with their expression was evaluated in the spinal cord and dorsal root ganglion (DRG). Nociception was assessed in the formalin, capsaicin, and acetic acid writhing tests. The expression of 5-HT5A receptors was determined by Western blot analysis. Intrathecal treatment with serotonin (5-HT, 10-100 nmol) or 5-carboxamidotryptamine (5-CT, 0.03-0.3 nmol) dose-dependently prevented 1% formalin-induced nociception. Furthermore, 5-HT reduced capsaicin- and acetic acid-induced nociception. 5-HT- or 5-CT-induced antinociception in the formalin test was diminished by the selective 5-HT5A receptor antagonist N-[2-(dimethylamino)ethyl]-N-[[4'-[[(2-phenylethyl)amino] methyl][1,1'-biphenyl]-4-yl]methyl]cyclopentanepropanamide dihydrochloride (SB-699551; 3 and 10 nmol). In addition, 5-HT-induced spinal antinociception in the capsaicin and acetic acid tests was blocked by SB-699551 (10 nmol). Given alone, intrathecal injection of SB-699551 did not affect nociception induced by any irritant. 5-HT5A receptors were expressed in the dorsal spinal cord and DRG, even though formalin injection increased after 24h 5-HT5A receptor expression only in the spinal cord. Data suggest that 5-HT and 5-CT produce antinociception by activation of spinal 5-HT5A receptors in both the spinal cord and DRG. Furthermore, our results suggest that spinal 5-HT5A receptors play an antinociceptive role in several pain models in rats. 5-HT5A receptors may provide a therapeutic target to develop analgesic drugs.

Role of peripheral and spinal 5-HT2B receptors in formalin-induced nociception.

In this study we assessed the role of local peripheral and spinal serotonin 2B (5-HT(2B)) receptors in rats submitted to the formalin test. For this, local peripheral ipsilateral, but not contralateral, administration of the highly selective 5-HT(2B) receptor antagonist 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyridine (RS-127445, 0.01-1 nmol/paw) significantly prevented 1% formalin-induced flinching behavior. Moreover, local peripheral ipsilateral, but not contralateral, of the selective 5-HT(2) receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI, 1-10 nmol/paw) augmented 0.5% formalin-induced nociceptive behavior. The local pronociceptive effect of the 5-HT(2) receptor agonist DOI (10 nmol/paw) was significantly prevented by the local injection of RS-127445 (0.01 nmol/paw). Moreover, intrathecal injection of the selective 5-HT(2B) receptor antagonist RS-127445 (0.1-10 nmol/rat) also prevented 1% formalin-induced nociceptive behavior. In contrast, spinal injection of the 5-HT(2) receptor agonist DOI (1-10 nmol/rat) significantly increased flinching behavior induced by 0.5% formalin. The spinal pronociceptive effect of the 5-HT(2) receptor agonist DOI (10 nmol/rat) was prevented by the intrathecal injection of the 5-HT(2B) receptor antagonist RS-127445 (0.1 nmol/rat). Our results suggest that the 5-HT(2B) receptors play a pronociceptive role in peripheral as well as spinal sites in the rat formalin test. 5-HT(2B) receptors could be a target to develop analgesic drugs.

Blockade of peripheral and spinal Na+/H+ exchanger increases formalin-induced long-lasting mechanical allodynia and hyperalgesia in rats.

The Na(+)/H(+) exchanger (NHE) is involved in the regulation of intracellular pH and volume by mediating the electroneutral transport of H(+) against an influx of Na(+) ions. Since NHE1 regulates pH in neurons and astrocytes and it is expressed in nociceptive nerve fibers, it is likely that NHE may modulate neuronal excitability and pain transmission. The purpose of this study was to assess the participation of peripheral and spinal NHE in the secondary allodynia/hyperalgesia induced by formalin. In addition, we determined whether formalin injection modifies the expression of NHE1 in lumbar dorsal root ganglia (DRG) and dorsal spinal cord. Subcutaneous injection of 0.5% formalin into the dorsal surface of the hind paw produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-lasting bilateral secondary mechanical allodynia/hyperalgesia. Peripheral and intrathecal pre-treatment (-10min) with selective NHE inhibitors 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.3-30µM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 0.3-30µM) and [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine dihydrochloride (zoniporide, 0.03-3µM) significantly increased 0.5% formalin-induced bilateral long-lasting secondary allodynia/hyperalgesia. Contrariwise, local peripheral or intrathecal post-treatment (day 6 postinjection) with these NHE inhibitors did not affect formalin-induced nociceptive behaviors. Formalin injection reduced NHE1 expression in ipsilateral and contralateral spinal dorsal horns from day 1 to 12. In addition, formalin diminished NHE1 protein expression in DRG at day 12. These results suggest that NHE1 plays a role in pain processing at peripheral and spinal levels in formalin-induced long-lasting nociceptive behaviors. Additionally, these results suggest that proteins involved in pH regulation could be targets for the development of new analgesic drugs.

Blockade of 5-HT7 receptors reduces tactile allodynia in the rat.

This study assessed the role of systemic and spinal 5-HT(7) receptors on rats submitted to spinal nerve injury. In addition, the 5-HT(7) receptors level in dorsal root ganglion and spinal cord was also determined. Tactile allodynia was induced by L5/L6 spinal nerve ligation. Systemic (0.01-10mg/kg) or spinal (0.3-30 µg) administration of the selective 5-HT(7) receptor antagonist SB-269970 but not vehicle reduced in a dose-dependent manner established tactile allodynia. This effect was maintained for about 6h. SB-269970 was more potent and effective by the spinal administration route than through systemic injection. Spinal nerve ligation reduced expression of 5-HT(7) receptors in the ipsilateral but not contralateral dorsal root ganglia. Moreover, 5-HT(7) receptor levels were lower in the ipsilateral dorsal spinal cord of neuropathic rats compared to naïve and sham rats. No changes in the receptor levels were observed in the contralateral dorsal spinal cord and in both regions of the ventral spinal cord. Data suggest that spinal 5-HT(7) receptors play a pronociceptive role in neuropathic rats. Results also indicate that spinal nerve injury leads to a reduced 5-HT(7) receptors level in pain processing-related areas which may result from its nociceptive role in this model. Data suggest that selective 5-HT(7) receptor antagonists may function as analgesics in nerve injury pain states.