Down Regulation of Catsper1 Expression by Calmodulin Inhibitor (Calmidazolium): Possible Implications for Fertility.

The CatSper channel localizes exclusively in the flagella of sperm cells. The Catsper1 protein, together with three pore units, is essential for the CatSper Channel formation, which produces flagellum hyperactivation and confers sperm fertility. Catsper1 expression is dependent on Sox transcription factors, which can recognize in vitro at least three Sox binding sites on the promoter. Sox transcription factors have calmodulin-binding domains for nuclear importation. Calmodulin (CaM) is affected by the specific inhibitor calmidazolium (CMZ), which prevents the nuclear transport of Sox factors. In this work, we assess the regulation of the Catsper1 promoter in vivo by Sox factors in the murine testis and evaluate the effects of the inhibitor calmidazolium on the expression of the Casper genes, and the motility and fertility of the sperm. Catsper1 promoter has significant transcriptional activity in vivo; on the contrary, three Sox site mutants in the Catsper1 promoter reduced transcriptional activity in the testis. CaM inhibition affects Sox factor nuclear transport and has notable implications in the expression and production of Catsper1, as well as in the motility and fertility capability of sperm. The molecular mechanism described here might conform to the basis of a male contraceptive strategy acting at the transcriptional level by affecting the production of the CatSper channel, a fundamental piece of male fertility.

PKC- and PKA-dependent phosphorylation modulates TREK-1 function in naïve and neuropathic rats.

PKC and PKA phosphorylation inhibit TREK-1 channels downstream of Gs protein-coupled receptor activation in vitro. However, the role of phosphorylation of TREK-1 in neuropathic pain is unknown. The purpose of this study was to investigate whether altered TREK-1 channel function by PKA and PKC modulators contributes to antiallodynia in neuropathic rats. Furthermore, we investigated if the in vitro described sites for PKC and PKA phosphorylation (S300 and S333, respectively) participate in the modulation of TREK-1 in naïve and neuropathic rats. L5/L6 spinal nerve ligation (SNL) induced tactile allodynia. Intrathecal injection of BL-1249 (TREK-1 activator) reversed nerve injury-induced tactile allodynia, whereas spadin (TREK-1 blocker) produced tactile allodynia in naïve rats and reversed the antiallodynic effect induced by BL-1249 in neuropathic rats. Intrathecal administration of rottlerin or Rp-cAMPs (PKC and PKA inhibitors, respectively) enhanced the antiallodynia observed with BL-1249 in neuropathic rats. In contrast, pretreatment with PdBu or forskolin (PKC and PKA activators, respectively) reduced the BL-1249-induced antiallodynia. Intrathecal injection of two high-activity TREK-1 recombinant channels, using a in vivo transfection method with lipofectamine, with mutations at PKC/PKA phosphosites (S300A and S333A) reversed tactile allodynia in neuropathic rats, with no effect in naïve rats. In contrast, transfection of two low-activity TREK-1 recombinant channels with phosphomimetic mutations at those sites (S300D and S333D) produced tactile allodynia in naïve rats and interfered with antiallodynic effects of rottlerin/BL-1249 or Rp-cAMPs/BL-1249. Data suggest that TREK-1 channel activity can be dynamically tuned in vivo by PKC/PKA to provoke allodynia and modulate its antiallodynic role in neuropathic pain.

Sex-dependent pronociceptive role of spinal α5 -GABAA receptor and its epigenetic regulation in neuropathic rodents.

Extrasynaptic α5 -subunit containing GABAA (α5 -GABAA ) receptors participate in chronic pain. Previously, we reported a sex difference in the action of α5 -GABAA receptors in dysfunctional pain. However, the underlying mechanisms remain unknown. The aim of this study was to examine this sexual dimorphism in neuropathic rodents and the mechanisms involved. Female and male Wistar rats or ICR mice were subjected to nerve injury followed by α5 -GABAA receptor inverse agonist intrathecal administration, L-655,708. The drug produced an antiallodynic effect in nerve-injured female rats and mice, and a lower effect in males. We hypothesized that changes in α5 -GABAA receptor, probably influenced by hormonal and epigenetic status, might underlie this sex difference. Thus, we performed qPCR and western blot. Nerve injury increased α5 -GABAA mRNA and protein in female dorsal root ganglia (DRG) and decreased them in DRG and spinal cord of males. To investigate the hormonal influence over α5 -GABAA receptor actions, we performed nerve injury to ovariectomized rats and reconstituted them with 17ß-estradiol (E2). Ovariectomy abrogated L-655,708 antiallodynic effect and E2 restored it. Ovariectomy decreased α5 -GABAA receptor and estrogen receptor α protein in DRG of neuropathic female rats, while E2 enhanced them. Since DNA methylation might contribute to α5 -GABAA receptor down-regulation in males, we examined CpG island DNA methylation of α5 -GABAA receptor coding gene through pyrosequencing. Nerve injury increased methylation in male, but not female rats. Pharmacological inhibition of DNA methyltransferases increased α5 -GABAA receptor and enabled L-655,708 antinociceptive effect in male rats. These results suggest that α5 -GABAA receptor is a suitable target to treat chronic pain in females.

Role of spinal P2Y6 and P2Y11 receptors in neuropathic pain in rats: possible involvement of glial cells.

BACKGROUND: The participation of spinal P2X receptors in neuropathic pain is well recognized. However, the role of P2Y receptors has been less studied. The purpose of this study was to investigate the contribution of spinal P2Y6,11 receptors following peripheral nerve damage induced by spinal nerve ligation. In addition, we determined the expression of P2Y6,11 receptors in the dorsal spinal cord in presence of the selective P2Y6,11 receptors antagonists. Furthermore, we evaluated the participation of spinal microglia and astrocytes in the pronociceptive role of P2Y6,11 receptors. RESULTS: Spinal administration of the selective P2Y6 (MRS2578, 10-100 µM) and P2Y11 (NF340, 0.3-30 µM) receptor antagonists reduced tactile allodynia in spinal nerve ligated rats. Nerve injury increased the expression of P2Y6,11 receptors at 7, 14 and 21 days after injury. Furthermore, intrathecal administration of MRS2578 (100 µM/day) and NF340 (30 µM/day) for 3 days significantly reduced spinal nerve injury-induced increase in P2Y6,11 receptors expression, respectively. Spinal treatment (on day 14 after injury) with minocycline (100 µg/day) or fluorocitrate (1 nmol/day) for 7 days reduced tactile allodynia and spinal nerve injury-induced up-regulation in Iba-1 and GFAP, respectively. In addition, minocycline reduced nerve injury-induced up-regulation in P2Y6,11 receptors whereas that fluorocitrate diminished P2Y11, but not P2Y6, receptors up-regulation. Intrathecal treatment (on day 21 after injury) with the selective P2Y6 (PSB0474, 3-30 µM) and P2Y11 (NF546, 1-10 µM) receptor agonists produced remarkable tactile allodynia in nerve ligated rats previously treated with minocycline or fluorocitrate for 7 days. CONCLUSIONS: Our data suggest that spinal P2Y6 is present in spinal microglia while P2Y11 receptors are present in both spinal microglia and astrocytes, and both receptors are up-regulated in rats subjected to spinal nerve injury. In addition, our data suggest that the spinal P2Y6 and P2Y11 receptors participate in the maintenance of neuropathic pain.

Estrogen receptor α regulates the IKKs/NF-kB activity involved in the development of mechanical allodynia induced by REM sleep deprivation in rats.

Several signaling pathways that converge in NF-kB activation have been linked to developing and maintaining different types of pathological pain. In addition, some mechanisms implied in the establishment of chronic pain have been demonstrated to have a sex-dependent correlation. This study aimed to determine if the IKKs/NF-kB signaling pathway is involved in establishing REM sleep deprivation (REMSD) induced mechanical allodynia in rats and its possible regulation depending on estradiol and estrogen receptors. Intrathecal administration of BMS-345541 or minocycline, two drugs that reduce the IKKs/NF-kB activity, avoided the development of mechanical allodynia in female but not in male rats subjected to 48 h of REMSD. Ovariectomy in female rats abolished the effect of BMS-345541 and minocycline. Meanwhile, the 17-ß-estradiol restitution restored it. Intrathecal administration of MPP, a selective ERα antagonist, but not PHTPP, a selective ERß antagonist, avoided the effect of BMS-345541 in female rats without hormonal manipulation. In addition, the transient run-down of ERα in female rats abolished the effect of BMS-345541. All data suggest an important role of ERα as a regulator of the IKKs/NF-kB activity. REMSD increased the ERα protein expression in the dorsal root ganglia and the dorsal spinal cord in females but not in male rats. Interestingly, ERα activation or ERα overexpression allowed the effect of BMS-345541 in male rats. Data suggest an important regulatory role of ERα in the IKKs/NF-kB activity on establishing mechanical allodynia induced by REMSD in female rats.

Chronic restraint stress and social transfer of stress produce tactile allodynia mediated by the HMGB1/TNFα/TNFR1 pathway in female and male rats.

Previous studies have shown the relevance of high mobility group box 1 protein (HMGB1) and tumor necrosis factor α (TNFα) in nerve or tissue injury-induced nociception. However, the role of these proteins in chronic stress and social transfer of stress (STS)-induced dysfunctional pain is not entirely known. The aim of this study was to determine the participation of the spinal HMGB1-TNFα signaling pathway and TNFα receptor 1 (TNFR1) in rats subjected to chronic restraint stress (CRS) and STS. Non-stressed female and male rats in contact with CRS rats increased sniffing behavior of the anogenital area, behavior related to STS. Rats subjected to CRS and STS reduced 50 % withdrawal threshold and reached the value of tactile allodynia after 21 days of stress. Rats return to the basal withdrawal threshold after 30 days without stress and return to allodynia values in only 5 days of stress sessions (priming). Female and male rats subjected to 28 days of CRS or STS were intrathecal injected with glycyrrhizin (inhibitor of HMGB1), thalidomide (inhibitor of the TNFα synthesis), and R7050 (TNFR1 antagonist), in all the cases, an antiallodynic effect was observed. Rats under CRS or STS enhanced HMGB1 and TNFR1 protein expression in DRG and dorsal spinal cord. Data suggest that the spinal HMGB1/TNFα/TNFR1 signaling pathway plays a relevant role in the maintenance of CRS and STS-induced nociceptive hypersensitivity in rats. These proteins could be helpful in developing pain treatments for fibromyalgia in humans.

Spinal bestrophin-1 and anoctamin-1 channels have a pronociceptive role in the tactile allodynia induced by REM sleep deprivation in rats.

Bestrophin-1 and anoctamin-1 are members of the calcium-activated chloride channels (CaCCs) family and are involved in inflammatory and neuropathic pain. However, their role in pain hypersensitivity induced by REM sleep deprivation (REMSD) has not been studied. This study aimed to determine if anoctamin-1 and bestrophin-1 are involved in the pain hypersensitivity induced by REMSD. We used the multiple-platform method to induce REMSD. REM sleep deprivation for 48 h induced tactile allodynia and a transient increase in corticosterone concentration at the beginning of the protocol (12 h) in female and male rats. REMSD enhanced c-Fos and α2δ-1 protein expression but did not change activating transcription factor 3 (ATF3) and KCC2 expression in dorsal root ganglia and dorsal spinal cord. Intrathecal injection of CaCCinh-A01, a non-selective bestrophin-1 blocker, and T16Ainh-A01, a specific anoctamin-1 blocker, reverted REMSD-induced tactile allodynia. However, T16Ainh-A01 had a higher antiallodynic effect in male than female rats. In addition, REMSD increased bestrophin-1 protein expression in DRG but not in DSC in male and female rats. In marked contrast, REMSD decreased anoctamin-1 protein expression in DSC but not in DRG, only in female rats. Bestrophin-1 and anoctamin-1 promote pain and maintain tactile allodynia induced by REM sleep deprivation in both male and female rats, but their expression patterns differ between the sexes.

The Estrogen Receptor Alpha Regulates the Sex-dependent Expression and Pronociceptive Role of Bestrophin-1 in Neuropathic Rats.

Bestrophin-1, a calcium-activated chloride channel (CaCC), is involved in neuropathic pain; however, it is unclear whether it has a dimorphic role in female and male neuropathic rats. This study investigated if 17ß-estradiol and estrogen receptor alpha (ERα) activation regulate bestrophin-1 activity and expression in neuropathic rats. Neuropathic pain was induced by L5-spinal nerve transection (SNT). Intrathecal administration of CaCCinh-A01 (.1-1 µg), a CaCC blocker, reversed tactile allodynia induced by SNT in female but not male rats. In contrast, T16Ainh-A01, a selective anoctamin-1 blocker, had an equal antiallodynic effect in both sexes. SNT increased bestrophin-1 protein expression in injured L5 dorsal root ganglia (DRG) in female rats but decreased bestrophin-1 protein in L5 DRG in male rats. Ovariectomy prevented the antiallodynic effect of CaCCinh-A01, but 17ß-estradiol replacement restored it. The effect of CaCCinh-A01 was prevented by intrathecal administration of MPP, a selective ERα antagonist, in rats with and without prior hormonal manipulation. In female rats with neuropathy, ovariectomy prevented the increase in bestrophin-1 and ERα protein expression, while 17ß-estradiol replacement allowed for an increase in both proteins in L5 DRG. Furthermore, ERα antagonism (with MPP) prevented the increase in bestrophin-1 and ERα protein expression. Finally, ERα activation with PPT, an ERα selective activator, induced the antiallodynic effect of CaCCinh-A01 in neuropathic male rats and prevented the reduction in bestrophin-1 protein expression in L5 DRG. In summary, data suggest ERα activation is necessary for bestrophin-1's pronociceptive action to maintain neuropathic pain in female rats. PERSPECTIVE: The mechanisms involved in neuropathic pain differ between male and female animals. Our data suggest that ERα is necessary for expression and function of bestrophin-1 in neuropathic female but not male rats. Data support the idea that a therapeutic approach to relieving neuropathic pain must be based on patient's gender.

Bestrophin-1 Participates in Neuropathic Pain Induced by Spinal Nerve Transection but not Spinal Nerve Ligation.

Previous studies have reported that L5/L6 spinal nerve ligation (SNL), but not L5 spinal nerve transection (SNT), enhances anoctamin-1 in injured and uninjured dorsal root ganglia (DRG) of rats suggesting some differences in function of the type of nerve injury. The role of bestrophin-1 in these conditions is unknown. The aim of this study was to investigate the role of bestrophin-1 in rats subjected to L5 SNT and L5/L6 SNL. SNT up-regulated bestrophin-1 protein expression in injured L5 and uninjured L4 DRG at day 7, whereas it enhanced GAP43 mainly in injured, but also in uninjured DRG. In contrast, SNL enhanced GAP43 at day 1 and 7, while bestrophin-1 expression increased only at day 1 after nerve injury. Accordingly, intrathecal injection of the bestrophin-1 blocker CaCCinh-A01 (1-10 µg) reverted SNT- or SNL-induced tactile allodynia in a concentration-dependent manner. Intrathecal injection of CaCCinh-A01 (10 µg) prevented SNT-induced upregulation of bestrophin-1 and GAP43 at day 7. In contrast, CaCCinh-A01 did not affect SNL-induced up-regulation of GAP43 nor bestrophin-1. Bestrophin-1 was mainly expressed in small- and medium-size neurons in naïve rats, while SNT increased bestrophin-1 immunoreactivity in CGRP+, but not in IB4+ neuronal cells in DRG. Intrathecal injection of bestrophin-1 plasmid (pCMVBest) induced tactile allodynia and increased bestrophin-1 expression in DRG and spinal cord in naïve rats. CaCCinh-A01 reversed bestrophin-1 overexpression-induced tactile allodynia and restored bestrophin-1 expression. Our data suggest that bestrophin-1 plays a relevant role in neuropathic pain induced by SNT, but not by SNL. PERSPECTIVE: SNT, but not SNL, up-regulates bestrophin-1 and GAP43 protein expression in injured L5 and uninjured L4 DRG. SNT increases bestrophin-1 immunoreactivity in CGRP+ neurons in DRG. Bestrophin-1 overexpression induces allodynia. CaCCinh-A01 reduces allodynia and restores bestrophin-1 expression. Our data suggest bestrophin-1 is differentially regulated depending on the neuropathic pain model.

Activation of α 6 -containing GABA A receptors induces antinociception under physiological and pathological conditions.

ABSTRACT: The loss of GABAergic inhibition is a mechanism that underlies neuropathic pain. Therefore, rescuing the GABAergic inhibitory tone through the activation of GABA A receptors is a strategy to reduce neuropathic pain. This study was designed to elucidate the function of the spinal α 6 -containing GABA A receptor in physiological conditions and neuropathic pain in female and male rats. Results show that α 6 -containing GABA A receptor blockade or transient α 6 -containing GABA A receptor knockdown induces evoked hypersensitivity and spontaneous pain in naive female rats. The α 6 subunit is expressed in IB4 + and CGRP + primary afferent neurons in the rat spinal dorsal horn and dorsal root ganglia but not astrocytes. Nerve injury reduces α 6 subunit protein expression in the central terminals of the primary afferent neurons and dorsal root ganglia, whereas intrathecal administration of positive allosteric modulators of the α 6 -containing GABA A receptor reduces tactile allodynia and spontaneous nociceptive behaviors in female, but not male, neuropathic rats and mice. Overexpression of the spinal α 6 subunit reduces tactile allodynia and restores α 6 subunit expression in neuropathic rats. Positive allosteric modulators of the α 6 -containing GABA A receptor induces a greater antiallodynic effect in female rats and mice compared with male rats and mice. Finally, α 6 subunit is expressed in humans. This receptor is found in CGRP + and P2X3 + primary afferent fibers but not astrocytes in the human spinal dorsal horn. Our results suggest that the spinal α 6 -containing GABA A receptor has a sex-specific antinociceptive role in neuropathic pain, suggesting that this receptor may represent an interesting target to develop a novel treatment for neuropathic pain.

Effect of chronic administration of 17ß-estradiol on the vasopressor responses induced by the sympathetic nervous system in insulin resistance rats.

Several studies have demonstrated that the underlying mechanism of insulin resistance (IR) is linked with developing diseases like diabetes mellitus, hypertension, metabolic syndrome, and polycystic ovary syndrome. In turn, the dysfunction of female gonadal hormones (especially 17ß-estradiol) may be related to the development of IR complications since different studies have shown that 17ß-estradiol has a cardioprotector and vasorelaxant effect. This study aimed was to determine the effect of the 17ß-estradiol administration in insulin-resistant rats and its effects on cardiovascular responses in pithed rats. Thus, the vasopressor responses are induced by sympathetic stimulation or i.v. bolus injections of noradrenaline (α1/2), methoxamine (α1), and UK 14,304 (α2) adrenergic agonist were determined in female pithed rats with fructose-induced insulin resistance or control rats treated with: 1) 17ß-estradiol or 2) its vehicle (oil) for 5 weeks. Thus, 17ß-estradiol decreased heart rate, prevented the increase of blood pressure induced by ovariectomy, but with the opposite effect on sham-operated rats; and decreased vasopressor responses induced by i.v. bolus injections of noradrenaline on sham-operated (control and fructose group) and ovariectomized (control) rats, and those induced by i.v. bolus injections of methoxamine (α1 adrenergic agonist). Overall, these results suggest 17ß-estradiol has a cardioprotective effect, and its effect on vasopressor responses could be mediated mainly by the α1 adrenergic receptor. In contrast, IR with ovariectomy 17ß-estradiol decreases or loses its cardioprotector effect, this could suggest a possible link between the adrenergic receptors and the insulin pathway.

TREK-1 potassium channels participate in acute and long-lasting nociceptive hypersensitivity induced by formalin in rats.

TREK-1 channels are expressed in small nociceptive dorsal root ganglion (DRG) neurons where they participate in acute inflammatory and neuropathic pain. However, the role of TREK-1 in persistent pain is not well understood. The aim of this study was to investigate the local peripheral and spinal participation of TREK-1 in formalin-induced acute and long-lasting nociceptive hypersensitivity. Local peripheral or intrathecal pre-treatment with spadin, selective blocker of TREK-1, increased acute flinching behavior and secondary mechanical allodynia and hyperalgesia behavior observed 6 days after formalin injection. Local peripheral or intrathecal pre-treatment with BL-1249, selective opener of TREK-1, decreased long-lasting secondary mechanical allodynia and hyperalgesia induced by formalin. Pre-treatment with BL-1249 prevented the pro-nociceptive effect of spadin on acute nociception and long-lasting mechanical allodynia and hyperalgesia in rats. Pre-treatment with two recombinant channels that produce a high TREK-1 current, S300A and S333A (non-phosphorylated state of TREK-1), reduced formalin-induced acute pain and long-lasting mechanical allodynia and hyperalgesia. Besides, post-treatment with S300A, S333A or BL-1249 reversed long-lasting mechanical allodynia and hyperalgesia induced by formalin. Formalin increased TREK-1 expression at 1 and 6 days in DRG and dorsal spinal cord in rats, whereas that it increased c-fos expression at the DRG. Intrathecal repeated transfection of rats with S300A and S333A or injection with BL-1249 reduced formalin-induced enhanced c-fos expression. Data suggest that TREK-1 activity at peripheral and spinal sites reduces neuronal excitability in the process of acute and long-lasting nociception induced by formalin in rats.

Interaction of NHE1 and TRPA1 Activity in DRG Neurons Isolated from Adult Rats and its Role in Inflammatory Nociception.

Transient receptor potential ankyrin 1 (TRPA1) channel is expressed in a subset of nociceptive neurons. This channel integrates several nociceptive signals. Particularly, it is modulated by intracellular pH (pHi). Na+/H+ exchanger 1 (NHE1) contributes to the maintenance of pHi in nociceptors. However, it is currently unknown whether the interaction between TRPA1 and NHE1 contributes to the nociceptive processing. Thus, the purpose of this study was to assess the functional interaction between NHE1 and TRPA1 in small dorsal root ganglion (DRG) neurons from primary culture obtained from adult rats. Moreover, we also evaluated their possible interaction in acute and inflammatory pain. Zoniporide (selective NHE1 inhibitor) reduced pHi and increased intracellular calcium in a concentration-dependent fashion in DRG neurons. Zoniporide and allyl isothiocyanate (AITC, TRPA1 agonist) increased calcium transients in the same DRG neuron, whereas that A-967079 (TRPA1 antagonist) prevented the effect of zoniporide in DRG neurons. Repeated AITC induced TRPA1 desensitization and this effect was prevented by zoniporide. Both NHE1 and TRPA1 were localized at the membrane surface of DRG neurons in culture. Local peripheral zoniporide enhanced AITC-induced pronociception and this effect was prevented by A-967079. Likewise, zoniporide potentiated Complete Freund's Adjuvant (CFA)-induced hypersensitivity, effect which was prevented by A-967079 in vivo. CFA paw injection increased TRPA1 and decresed NHE1 protein expression in DRG. These results suggest a functional interaction between NHE1 and TRPA1 in DRG neurons in vitro. Moreover, data suggest that this interaction participates in acute and inflamatory pain conditions in vivo.

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.

Metformin: A Prospective Alternative for the Treatment of Chronic Pain.

Metformin (biguanide) is a drug widely used for the treatment of type 2 diabetes. This drug has been used for 60 years as a highly effective antihyperglycemic agent. The search for the mechanism of action of metformin has produced an enormous amount of research to explain its effects on gluconeogenesis, protein metabolism, fatty acid oxidation, oxidative stress, glucose uptake, autophagy and pain, among others. It was only up the end of the 1990s and beginning of this century that some of its mechanisms were revealed. Metformin induces its beneficial effects in diabetes through the activation of a master switch kinase named AMP-activated protein kinase (AMPK). Two upstream kinases account for the physiological activation of AMPK: liver kinase B1 and calcium/calmodulin-dependent protein kinase kinase 2. Once activated, AMPK inhibits the mechanistic target of rapamycin complex 1 (mTORC1), which in turn avoids the phosphorylation of p70 ribosomal protein S6 kinase 1 and phosphatidylinositol 3-kinase/protein kinase B signaling pathways and reduces cap-dependent translation initiation. Since metformin is a disease-modifying drug in type 2 diabetes, which reduces the mTORC1 signaling to induce its effects on neuronal plasticity, it was proposed that these mechanisms could also explain the antinociceptive effect of this drug in several models of chronic pain. These studies have highlighted the efficacy of this drug in chronic pain, such as that from neuropathy, insulin resistance, diabetic neuropathy, and fibromyalgia-type pain. Mounting evidence indicates that chronic pain may induce anxiety, depression and cognitive impairment in rodents and humans. Interestingly, metformin is able to reverse some of these consequences of pathological pain in rodents. The purpose of this review was to analyze the current evidence about the effects of metformin in chronic pain and three of its comorbidities (anxiety, depression and cognitive impairment).

Fructose-Induced Insulin Resistance as a Model of Neuropathic Pain in Rats.

Peripheral neuropathy is one of the main complications of diabetes. The pathogenesis of this affectation is not completely understood. Several studies refer to hyperglycemia as the principal cause of diabetic neuropathy. Nonetheless, there are changes in the expression of insulin receptor during the progress of diabetic neuropathy, suggesting that this disorder begins before high glucose blood levels are established. In this study, we investigated fructose-induced insulin resistance as a model of neuropathic pain. Insulin resistance was induced by 15% fructose in drinking water for 16 weeks. Fructose slightly enhanced blood glucose levels. In contrast, chronic fructose increased insulin plasma levels and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index. Moreover, fructose induced hyperalgesia (to 0.5% formalin) and tactile allodynia. Interestingly, gabapentin and metformin, but not diclofenac, reversed in a dose-dependent manner fructose-induced tactile allodynia. Fructose enhanced activating factor transcription 3 (ATF3), but not caspase-3 and α2δ-1 subunit, in individual L4 and L5 dorsal root ganglia (DRG) and sciatic nerve. Chronic fructose also increased anoctamin-1 and ASIC3 whereas it reduced insulin receptor-ß, α5GABAA receptors and TASK-3 channels protein expression in DRG and sciatic nerve. In contrast, fructose did not change TRPV1 channel protein expression. Treatment with metformin for 4 weeks reversed some of the fructose-induced changes in protein expression. Taken together, these data suggest that insulin resistance induced by fructose reproduces several aspects of neuropathic-like pain. Our data also suggest that nociceptive hypersensitivity in this model is due to the modulation of several ionic channels at the primary afferent neurons.

Spinal TASK-1 and TASK-3 modulate inflammatory and neuropathic pain.

This study assessed the participation of spinal TWIK-related acid-sensitive K+ channels 1 and 3 (TASK-1 and TASK-3) in inflammatory (formalin test) and neuropathic (spinal nerve ligation, SNL) pain in rats. Intrathecal pre-treatment (-10 min) with the TASK-1 blocker ML365 or TASK-3 blocker PK-THPP, but not vehicle, enhanced in a dose-dependent manner 1% formalin-induced acute and long-lasting secondary mechanical allodynia and mechanical hyperalgesia in rats. In contrast, intrathecal pre-treatment with terbinafine, an activator of TASK-3, reduced formalin-induced flinching and allodynia/hyperalgesia. Both blockers and terbinafine had similar effects on female and male rats. In addition, intrathecal injection of ML365 or PK-THPP blocked the terbinafine-induced antiallodynic effect in neuropathic rats, but they did not modify baseline withdrawal threshold in naïve or sham-operated rats. TASK-1 and TASK-3 mRNA and protein were expressed in L4 and L5 dorsal root ganglia (DRG) and dorsal and ventral spinal cord of naïve animals. Interestingly, formalin injection increased TASK-1 expression in ipsilateral L5 DRG, but not in the spinal cord. Moreover, formalin injection transiently enhanced TASK-3 expression in ipsilateral L5 DRG and dorsal spinal cord. In contrast, SNL down-regulated TASK-3 expression in the ipsilateral L4 and L5 DRG but not in dorsal or ventral spinal cord, while SNL did not modify TASK-1 expression at any tissue. The pharmacological and molecular results suggest that TASK-1 and TASK-3 have a relevant antinociceptive role in inflammatory and neuropathic pain.

Blockade of anoctamin-1 in injured and uninjured nerves reduces neuropathic pain.

The aim of this study was to determine the participation of anoctamin-1 in 2 models of neuropathic pain in rats (L5/L6 spinal nerve ligation [SNL] and L5 spinal nerve transection [SNT]). SNL and SNT diminished withdrawal threshold in rats. Moreover, SNL up-regulated anoctamin-1 protein expression in injured L5 and uninjured L4 DRG whereas that it enhanced activating transcription factor 3 (ATF-3) and caspase-3 expression only in injured L5 DRG. In marked contrast, SNT enhanced ATF-3 and caspase-3, but not anoctamin-1, expression in injured L5 DRG but it did not modify anoctamin-1, ATF-3 nor caspase-3 expression in uninjured L4 DRG. Accordingly, repeated (3 times) intrathecal injection of the anoctamin-1 blocker T16Ainh-A01 (0.1-1 µg) or MONNA (1-10 µg) partially reverted SNL-induced mechanical allodynia in a dose-dependent manner. In contrast, anoctamin-1 blockers only produced a modest effect in SNT-induced mechanical allodynia. Interestingly, intrathecal injection of T16Ainh-A01 (1 µg) or MONNA (10 µg) prevented SNL-induced up-regulation of anoctamin-1, ATF-3 and caspase-3 in injured L5 DRG. Repeated intrathecal injection of T16Ainh-A01 or MONNA also reduced SNT-induced up-regulation of ATF-3 in injured L5 DRG. In contrast, T16Ainh-A01 and MONNA did not affect SNT-induced up-regulation of caspase-3 expression in L5 DRG. Likewise, gabapentin (100 µg) diminished SNL-induced up-regulation of anoctamin-1, ATF-3 and caspase-3 expression in injured L5 DRG. These data suggest that spinal anoctamin-1 in injured and uninjured DRG participates in the maintenance of neuropathic pain in rats. Our data also indicate that expression of anoctamin-1 in DRG is differentially regulated depending on the neuropathic pain model.

Peripheral and spinal TRPA1 channels contribute to formalin-induced long-lasting mechanical hypersensitivity.

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.

Predominant role of spinal P2Y1 receptors in the development of neuropathic pain in rats.

The role of P2X2/3, P2X3, P2X4 or P2X7 and P2Y2, P2Y6, and P2Y12 receptors in neuropathic pain has been widely studied. In contrast, the role of P2Y1 receptors is scarcely studied. In this study we assessed the role of P2Y1 receptors in several neuropathic pain models in the rat. Furthermore, we analyzed the expression of P2Y1 receptors in the ipsilateral dorsal root ganglia (DRG) and dorsal part of the spinal cord during the development and maintenance of neuropathic pain. We also determined the effect of the P2Y1 receptor antagonist on the expression of P2Y1 receptors. Chronic constriction injury (CCI), spared nerve injury (SNI) or spinal nerve ligation (SNL) produced tactile allodynia from 1 to 14 days after nerve injury. CCI, SNI and SNL enhanced expression of P2Y1 receptors in DRG but not in the dorsal part of the spinal cord at 1-3 days after injury. Intrathecal injection of the selective P2Y1 receptor antagonist MRS2500, but not vehicle, reduced tactile allodynia in rats 1-3 days after CCI, SNI, or SNL. Moreover, intrathecal injection of MRS2500 (at day 1 or 3) reduced neuropathy-induced up-regulation of P2Y1 receptors expression. Intrathecal injection of MRS2500 lost most of the antiallodynic effect when injected 14 days after injury. At this time, MRS2500 did not modify nerve-injury-induced P2Y1 receptors up-regulation. Our results suggest that P2Y1 receptors are localized in DRG, are up-regulated by nerve injury and play a pronociceptive role in development and, to a lesser extent, maintenance of neuropathic pain.