GABA-Dependent Pain FAcilitation of Spinal 5-HT3R in Diabetic Neuropathic Pain

2014/2015 Daniel Artur Abreu Martins GABA-Dependent Pain Facilitation of Spinal 5-HT3R In Diabetic Neuropathic Pain março, 2015 Mestrado Integrado em Medicina Área: Neurociências Trabalho efetuado sob a Orientação de: Doutora Carla Sofia Costa Morgado E sob a Coorientação de: Doutora Isaura Ferreira Tavares Trabalho organizado de acordo com as normas da revista: European Journal of Pain Daniel Artur Abreu Martins GABA-Dependent Pain Facilitation of Spinal 5-HT3R in Diabetic Neuropathic Pain março, 2015 À"Professora"Doutora"Carla"Morgado," por"tudo"quanto"me"ensinou"e"permitiu"que"aprendesse"sozinho." " À""Professora"Doutora"Isaura"Tavares," pelas"palavras"de"encorajamento"e"pelos"desafios"constantes." " À"Mestre"Marta"Silva," a"companheira"de"todas"as"horas." " A"todos"os"elementos"do"Departamento"de"Biologia"Experimental," uma"segunda"família." ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! " " À"minha"família," pelo"suporte"e"dedicação." " À"Nádia," pelas"muitas"conversas"de"janela." " A"todos"os"meus"amigos," pelo"carinho,"companheirismo"e"gargalhadas." ! ! 1 GABA-Dependent Pain Facilitation of Spinal 5-HT3R In Diabetic Neuropathic Pain Running title: 5-HT3R pronociception in diabetic neuropathic pain M Silva*, D Martins*, I Tavares, C Morgado Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal Departamento de Biologia Experimental, Faculdade de Medicina do Porto, Universidade do Porto, Portugal * The authors contributed equally to the manuscript Corresponding author: Carla Morgado Department of Experimental Biology Faculty of Medicine of Porto Alameda Prof. Hernâni Monteiro 4200-319 Porto Portugal Fax: +351225513655 Phone: +351225513654 e-mail: [email protected] Category of manuscript original article Funding sources Supported by FPF7 EU Project REDDSTAR (FP7-Health-2012.2.4.3-1) Conflict of interests The authors declare no conflict of interests 2 What's already known about this topic?  Activation of 5-HT3 receptors expressed in GABAergic neurons of the spinal cord increases GABA release  Inhibition of spinal 5-HT3R decreases pain hypersensitivity in animal models of chronic inflammatory and traumatic neuropathic pain What does this study add?  Spinal 5-HT3 receptors facilitate pain transmission and contributes to ERKs-mediated spinal sensitization during diabetic neuropathy  5-HT3R-mediated pain facilitation during DNP is dependent on spinal GABAergic post-synaptic neurotransmission 3 Abstract Background: Spinal 5-HT3 receptors (5-HT3R) has been implicated in chronic pain development. The extent to which 5-HT3R contributes to spinal sensitization and pain during diabetic neuropathy (DN) remain elusive and the mechanisms subserving the effects of 5-HT3R activation on spinal pain processing are still unclear. This study aimed to evaluate the contribution of spinal 5-HT3R to pain facilitation and spinal sensitization during DN. Moreover, considering the pain facilitation mediated by spinal GABA in DN and the increased release of GABA upon 5-HT3R activation, the role of GABA as a mediator of 5-HT3R spinal effect was assessed. Methods: Mechanical nociception was evaluated by paw pressure test in streptozotocin (STZ)-diabetic and control rats after intrathecal (i.t.) administration of 5-HT3R antagonist (Y25130). The spinal activation of extracellular signal-regulated kinases (ERKs) pathway and the expression of 5-HT3R, glial fibrillary acidic protein (GFAP; marker of astroglia activation) and ionized calcium binding adaptor molecule 1 (IBA-1; marker of microglia activation) were evaluated at the peak maximum effect of Y25130. The involvement of GABA in the behavioural pain effect of Y25130, was assessed in STZ-diabetic animals receiving i.t administrations of muscimol (GABAAR agonist). Results: Intrathecal administration of Y25130 reverted mechanical hyperalgesia and ERK-mediated spinal sensitization in STZ-diabetic rats, while no effects were observed in control animals. The spinal activation of GABAAR by i.t administration of muscimol abolished Y25130-driven antinociception. The expression of IBA-1, GFAP and 5-HT3R was unaltered by treatment. Conclusion: These findings point for a GABA-dependent pronociceptive role of spinal 5-HT3R in this chronic pain condition. Key-Words: Diabetic neuropathic pain; 5-HT3R; GABA; Pain facilitation; ERK1/2, spinal sensitization 4 1. Introduction Diabetic neuropathic pain (DNP) is a debilitating complication of diabetes characterized by spontaneous pain, mechanical hyperalgesia and tactile allodynia (Galer et al., 2000). DNP have been mostly attributed to damage of peripheral nerves (Chen and Levine, 2001), however several studies have showed that functional impairments in spinal nociceptive processing account for pain during diabetes (Pertovaara et al., 2001; Chen and Pan, 2002; Morgado and Tavares, 2007; Morgado et al., 2010). Functional studies using the streptozotocin (STZ)-diabetic rat showed that impaired pain responses are accompanied by spontaneous hyperactivity and hyperexcitability of nociceptive spinal circuits (Morgado and Tavares, 2007; Li et al., 2010). These changes have been attributed to increased peripheral input and recruitment of nociceptive ascending pathways (Burchiel et al., 1985; Chen and Pan, 2002), to alterations of spinal nociceptive modulatory mechanisms (Morgado et al., 2008) and, more recently, to impairments in pain modulation from supraspinal areas (Paulson et al., 2007; Morgado et al., 2011b; Silva et al., 2013). Recent studies showed that the behavioral hypersensitivity and spinal neuronal hyperexcitability are accompanied by persistent activation of descending pain circuits (Morgado et al., 2011b; Silva et al., 2013), namely the descending serotoninergic pathways arising from the rostroventromedial medulla (RVM) (Morgado et al., 2011b). The RVM is a key brainstem relay station of the descending pain modulatory circuits, which modulates spinal pain transmission mainly by the release of serotonin. Depending on the receptor subtype activated and the pain condition serotonin can inhibit or enhance spinal nociceptive transmission (Dogrul et al., 2009). The 5-HT3 receptors (5-HT3R), the only 5-HT ionotropic receptor with excitatory functions, are expressed in the spinal dorsal horn neurons and in the central terminals of primary afferents (Kia et al., 1995). Experimental data on 5-HT3R-mediated modulation of spinal nociceptive processing during acute pain seem conflicting, with studies dividing between anti- and pronociceptive roles (Alhaider et al., 1991; Guo et al., 2014). A pain facilitatory role of spinal 5-HT3R has been demonstrated in animal models of chronic inflammatory and traumatic neuropathic pain (Rahman et al., 2009; 11 followed by Tukey post-hoc test for multiple comparisons. Independent sample t-test was used to compare metabolic parameters and pretreatment behavioral data between STZ and control animals. Statistical significance was settled at p < 0.05. Results are expressed as mean ± standard error of the mean (s.e.m.). 3. Results 3.1. Metabolic characterization Four weeks after the induction of diabetes, STZ-diabetic rats presented significantly increased blood glucose concentration (STZ: 512.19 ± 10.15 mg/dL; control: 121.3 ± 7.36 mg/dL; p < 0.0001) and hemoglobin A1C levels (STZ: 12.1 ± 0.25%; control: 4.8 ± 0.06% p < 0.0001), along with decreased body weights (STZ: 261.5 ± 6.55 g; control: 402.1 ± 5.98 g; p < 0.0001), when compared with age-matched control animals (Table S1), which is in accordance with previous reports using the same animal model (Courteix et al., 1993; Calcutt, 2004; Morgado and Tavares, 2007) and support the installation of diabetes. 3.2. Effect of i.t. administration of 5-HT3R antagonist on mechanical nociception Four weeks after diabetes onset, STZ-diabetic rats developed mechanical hyperalgesia (Fig 1) , as demonstrated by the significantly lower pretreatment paw withdrawal threshold (PWT) of STZ-diabetic animals when compared with control animals (STZ: 59.8 ± 3.77 g; control: 103.0 ± 2.49 g; p < 0.0001). Intrathecal delivery of Y25130 significantly increased the PWT in STZ-diabetic rats, showing an antinociceptive effect of Y25130 during DNP. The antinociceptive effects of Y25130 in STZ-diabetic animals were evident at 0.5h (STZ+Saline: 59.8 ± 5.05 g; STZ+Y25130: 88.4 ± 2.26 g; p < 0.001) and had a maximum peak at 4h (STZ+Saline: 61.0 ± 3.22 g; STZ+Y25130: 109.8 ± 7.39 g; p < 0.0001), returning to pretreatment values 6h post-administration (STZ+Y25130 pretreatment: 62.2 ± 3.38 g; STZ+Y25130: 71.4 ± 3.29 g; p > 12 0.05). The administration of Y25130 had no effect in the mechanical response thresholds of control rats in any time-point evaluated (control+Y25130 pretreatment: 101.8 ± 4.80 g; control+Y25130 at 0.5h post-injection: 96.6 ± 9.22 g, control+Y25130 at 4h post-injection: 102.8 ± 6.62, control+Y25130 at 6h post-injection: 95.2 ± 4.55 g g; p>0.05) (Fig 1). 3.3. Effect of i.t. administration of 5-HT3R antagonist on spinal ERK1/2 activation The STZ-diabetic rats receiving saline infusions presented a significantly higher number of pERK1/2- IR cells when compared with control animals (STZ+Saline: 657.9 ± 37.42; control+Saline: 306.9 ± 58.63, p < 0.01) (Fig 2). Administration of Y25130 to STZ-diabetic animals significantly reduced spinal ERK1/2 activation (Fig 2), as demonstrated by the significantly lower number of pERK1/2-IR cells observed in the spinal sections of Y25130-treated STZ- animals (STZ+Saline: 657.9 ± 37.42; STZ+Y25130: 417.0 ± 41.27, p < 0.05) (Fig 2a and c-f). The treatment had no effects in the number of pERK1/2-IR cells in the spinal cord of control rats (control+Saline: 306.9 ± 58.63; control+Y25130: 293.1 ± 44.67, p > 0.05). Western blotting quantification of pERK1/2 and ERK1/2 expression in spinal homogenates from STZ-diabetic and control animals treated with saline or Y25130 also showed that administration of Y25130 significantly reduced spinal ERK1/2 activation in STZ-diabetic rats, as demonstrated by the significantly lower phosphorylated fraction of ERK1/2 content in STZ-diabetic rats treated with Y25130 (STZ+Saline: 4.0 ± 1.00; STZ+Y25130: 1.2 ± 0.49; control+Saline: 1.5 ± 0.05; control+Y25130: 1.2 ± 0.34, p <0.05) (Fig 2b). 3.4. Effect of i.t. administration of 5-HT3R antagonist on spinal glia activation In order to evaluate if spinal 5-HT3R antagonism during DNP interferes with glia activity, we evaluate the effect of spinal 5-HT3R inhibition on spinal GFAP and IBA-1 expression levels. The expression of GFAP was significantly lower in STZ-diabetic rats than in controls animals (STZ+Saline: 0.04 ± 0.012; 13 control+Saline: 0.92 ± 0.085; p <0.01) (Fig S1b and c). The expression levels of IBA-1 were significantly higher in STZ-animals when compared with control rats (STZ+Saline: 1.9 ± 0.08; control+Saline: 1.1 ± 0.05; p <0.01) (Fig S1a and c). The spinal 5-HT3R inhibition did not affect the expression levels of GFAP (STZ+Saline: 0.04 ± 0.012; STZ+Y25130: 0.04 ± 0.006; p > 0.05; control+Saline: 0.92 ± 0.085; control+Y25130: 0.96 ± 0.21, p >0.05) and IBA- 1 (STZ+Saline: 1.9 ± 0.08; STZ+Y25130: 1.8 ± 0.12; p >0.05; control+Saline: 1.1 ± 0.05; control+Y25130: 1.2 ± 0.12; p >0.05), neither in STZ-diabetic nor in control rats (Fig S1). 3.5. Expression of 5-HT3RA at the spinal dorsal horn Since 5-HT3R inhibition had differential effects on nociceptive responses of STZ-diabetic and control animals, we hypothesized that the selective 5- HT3R-mediated pain facilitation during DNP could be explained by a possible change in the expression of the receptor induced by diabetes. Densitometric immunolabelling analysis of spinal 5-HT3RA expression did not reveal significant differences between the STZ-diabetic and control rats (STZ+Saline: 46.0 ± 1.90; STZ+Y25130: 39.8 ± 2.92; control+Saline: 40.6 ± 4.77; control+Y25130: 43.6 ± 2.94; p > 0.05) (Fig S2a and c-f). These findings were corroborated by western blotting analysis of the receptor expression (STZ+Saline: 0.3 ± 0.08; STZ+Y25130: 0.5 ± 0.14; control+Saline: 0.3 ± 0.02; control+Y25130: 0.4 ± 0.12; p > 0.05) (Fig S2b). 3.6. Effect of i.t. administration of GABAAR agonist on the effect of spinal 5-HT3R inhibition Muscimol administration prevented the antinociceptive effect elicited by 5-HT3R inhibition in STZ-diabetic rats, as showed by the reduction in PWT to levels of pretreatment in muscimol+Y25130-treated STZ-animals (STZ+Y25130 pretreatment: 60.6 ± 2.46 g; STZ+Y25130 at 4h post-injection: 109.8 ± 7.39 g; STZ+Y25130+Muscimol at 4h post-injection: 57.0 ± 6.43 g, p<0.001 in STZ+Y25130 at 4h post-injection vs the other groups) (Fig 3). Muscimol had no effects in the mechanical response thresholds of STZ- 14 diabetic animals receiving saline infusions (STZ+Saline pretreatment: 61.0 ± 3.22 g; STZ+Saline+Muscimol at 4h post-saline injection: 56.0 ± 7.10 g, p>0.05) (Fig 3). Discussion By using a pharmacological approach to inhibit the 5-HT3R at the spinal cord in a validated model of DNP, the present study is the first to demonstrate that spinal 5-HT3R activation is involved in pain facilitation and contributes to spinal sensitization through the activation of ERK1/2 pathways during DNP. The present study also provides new insights into the mechanisms underlying 5-HT3R-spinal nociceptive modulation in chronic pain by showing that 5-HT3R pronociception is mediated by spinal GABAergic signalling. The development of persistent pain appears to be dependent, in part, upon increased drive of RVM-arising 5-HT descending pathways, which leading to activation of 5-HT3Rs at the spinal level, seems to facilitate pain transmission during chronic pain (Suzuki et al., 2002; Dogrul et al., 2009). The pain behavior detected in the second-phase of the formalin test, but not that observed in the first-phase, has been reported to be significantly reduced in mice lacking the subunit A of 5-HT3R and in animals receiving intrathecal administrations of 5-HT3R antagonists, suggesting an involvement of 5-HT3R in pain chronification (Oyama et al., 1996; Zeitz et al., 2002). However, some contradictory evidences exist in what concern the role of spinal 5-HT3R in pain modulation, with studies demonstrating that spinal 5-HT3R elicits antinociceptive effects in acute pain (Glaum et al., 1990; Alhaider et al., 1991). These findings point for a possible shift in the role of spinal 5-HT3R in chronic pain conditions. Our data show that inhibition of spinal 5-HT3R by intrathecal administration of a selective 5-HT3R antagonist reverted the mechanical hyperalgesia in STZ-diabetic rats. Previous studies using STZ- diabetic rats reported an increased activation of serotonergic neurons at the RVM along with higher spinal serotonin contents during DNP (Morgado et al., 2011b), suggesting an increased RVM descending serotoninergic drive during this chronic pain condition. This may lead to overactivation of spinal 5-HT3R, 15 which, taking into account the pain facilitatory role here reported, is likely to contribute to mechanical hypersensitivity associated to DNP. No differences were observed in the spinal expression of 5-HT3RA in STZ-diabetic rats, which reinforces the hypothesis that 5-HT3R-mediated pain facilitation during DNP is likely to be caused by increased serotonin bioavailability rather than due to changes in the expression of the receptor. Studies also showed unaltered 5-HT3R expression in other chronic pain conditions (Rahman et al., 2009). Our findings along with the lack of antinociceptive effect of a 5-HT3R antagonist in an animal model of traumatic neuropathy, where increased descending serotoninergic drive was not verified (Peters et al., 2010), clearly point to the important role of enhanced activity of descending serotoninergic pathways in spinal 5-HT3R pain facilitation during DNP. Increasing evidences show that extracellular signal-regulated kinases 1 and 2 (ERK1/2) expressed in the spinal cord are involved in nociceptive processing and spinal sensitization (Gao and Ji, 2009; Han et al., 2011). In fact, ERK1/2 are strongly activated in the spinal dorsal horn following peripheral inflammation and tissue/nerve injury and the pharmacological blockade of this activation reduces the hypersensitivity otherwise observed in these experimental models (Ji et al., 1999; 2002). This activation of ERKs has been attributed to the hyperexcitatibility of spinal dorsal horn neurons evoked by increased peripheral barrage. This assertion, while reasonable, discards the possible contribution of other dorsal horn inputs that are believed to regulate dorsal horn excitability, namely the inputs arising from descending modulatory pathways. Increased activation of ERK1/2 in STZ-diabetic rats was observed in the present study, in agreement with data from a previous study (Daulhac et al., 2006). In addition, our findings demonstrate that the i.t. administration of 5-HT3R antagonist reverted the increased spinal ERK1/2 activation in STZ-diabetic rats, pointing for a role of 5-HT3R-mediated descending serotoninergic facilitation in spinal sensitization during DNP. Consistent with our data, previous studies demonstrated that the depletion of spinal 5-HT reduced the formalin evoked flinching and activation of spinal ERK1/2 pathways (Svensson et al., 2006). Moreover, intrathecal administration of ondansetron (a 5-HT3 receptor antagonist) at doses that inhibited formalin-induced flinching also attenuated spinal ERK activation 16 (Svensson et al., 2006). Taken together these findings revealed that spinal ERKs activation requires the input from an excitatory serotoninergic pathway and is dependent on 5HT3R activity. At the spinal cord, 5-HT3R expression seems to be restricted to some neuronal subpopulations and primary afferents terminals (Kia et al., 1995), with no reported expression in glial cells (Guo et al., 2014). The mechanisms underlying 5-HT3R-mediated spinal pain modulation remain elusive. Recently, a 5-HT3R-dependent neuronal-glial crosstalk was proposed as a possible mechanism underlying pain facilitation induced by the pharmacological activation of spinal 5-HT3R in healthy animals (Guo et al., 2014), with an i.t. injection of 5-HT3R agonist leading to increased activation of microglia and astroglia and the reversion of these effect by treatment with a 5-HT3R specific antagonist. In the present study no differences were detected in the activation of spinal microglia or astroglia upon i.t. administration of 5-HT3R antagonist in STZ-diabetic rats. Our results do not seem to support a role for glia in the pronociceptive effects of spinal 5-HT3R, at least in this chronic pain condition. Indeed, it is important to note that Guo et al. (2014) studied the role of glial cells in the effects of spinal 5-HT3R activation in an acute pain condition and no reports exist on the effects of chronic pain in this mechanism. In the present study we used an animal model of chronic pain that already presents altered spinal glial responses before i.t. administration of 5-HT3R antagonist (Daulhac et al., 2006; Tsuda et al., 2008; Wodarski et al., 2009; Morgado et al., 2011a). The absence of a normal functioning of glia signalling cascade during DNP can contribute to the lack of effects of 5-HT3R on glia activation here reported. The interplay between 5-HT and GABA was shown to be crucial in spinal modulation of nociceptive transmission. The electrical stimulation of RVM neurons elicits GABA-mediated inhibitory post-synaptic potentials (IPSPs) in primate spinothalamic tract neurons (Giesler et al., 1981). Furthermore, the GABAA-R agonist, muscimol, and 2-methyl 5-HT, a nonselective 5-HTR agonist, was shown to have similar effects on nociceptive spinal projection neurons firing elicited by excitatory amino acids (Lei and Wilcox, 1990). More recently, 5-HT3R expression was reported in an intrinsic GABAergic neuronal subpopulation of the spinal cord, and its activation on 17 these neurons was shown to enhance GABA release (Fukushima et al., 2009). This enhancement is likely to activate postsynaptic GABAAR, which was shown to culminate in antinociception by inhibiting spinothalamic tract ascending neurons (Alhaider et al., 1991; Kawamata et al., 2003). If in normal conditions GABA seems to play a major inhibitory tone on spinal nociceptive transmission, several studies have been showing that persistent pain conditions, including DNP, are accompanied by a shift in the role of GABA from inhibitory to excitatory, due to the decrease of spinal potassium chloride co-transporter 2 (KCC2) expression (Jolivalt et al., 2008; Morgado et al., 2008). The KCC2 downregulation promotes an accumulation of intracellular chloride which causes an outflow of chloride ions upon the binding of GABA to GABAAR, leading to neuronal excitation instead of inhibition and contributing to the spinal nociceptive hyperactivity observed in DNP (Morgado et al., 2011a). Our results show that the antinociceptive effects of i.t. administered 5- HT3R antagonist is abolished by i.t. delivery of muscimol, suggesting that the effects elicited by 5-HT3R antagonism are mediated by the reduction of GABAAR activation, probably due to a decrease in spinal GABA release. In conclusion, the data gathered by the present study suggest that 5- HT3R-mediated increase in spinal GABAergic transmission, probably due to the overactivation of descending serotoninergic pathways, can mediate pain facilitation during DNP. This study provides new insights on the mechanisms underlying the contribution of spinal 5-HT3Rs to pain modulation during chronic pain. Accordingly, and attending to the already reported good tolerability and pharmacokinetic profile of 5-HT3Rs inhibitory drugs in the clinical practice (McCleane et al., 2003), the use of 5-HT3R antagonists may then be considered as a promising pharmacological approach in alleviating the mechanical hyperalgesia associated to diabetic neuropathy. Author contributions All authors participate in experimental conception and design. C. Morgado and I. Tavares supervised the experiments. M. Silva performed all the surgeries and behavioural tests. M. Silva and D. Martins performed 18 immunohistochemistry and western blot experiments. All authors discussed the results. M. Silva and D. Martins wrote the first drafts of the manuscript. C. Morgado and I. Tavares revised the manuscript. All authors have read and approved the final manuscript. References Alhaider, A. A., Lei, S. Z., and Wilcox, G. L. (1991). Spinal 5HT, Receptormediated Antinociception: Possible Release of GABA. The Journal of Neuroscience 11, 1881–1888. Brahmachari, S., Fung, Y. K., and Pahan, K. (2006). Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide. The Journal of Neuroscience 26, 4930–4939. Burchiel, K. J., Russell, L. C., Lee, R. P., and Sima, A. A. F. (1985). Spontaneous Activity of Primary Afferent Neurons in Diabetic BB/Wistar Rats. A Possible Mechanism of Chronic Diabetic Neuropathic Pain. Diabetes 34, 1210–1213. Calcutt, N. A. (2004). Modeling Diabetic Sensory Neuropathy in Rats. In Methods in Molecular Medicine, Vol 99: Pain Research: Methods and Protocols, Z. D. Luo, ed. (Humana Press Inc) pp. 1–12. Chen, S.-R., and Pan, H.-L. (2002). Hypersensitivity of Spinothalamic Tract Neurons Associated With Diabetic Neuropathic Pain in Rats. Journal of Neurophysiology 87, 2726–2733. Chen, X., and Levine, J. D. (2001). Hyper-responsivity in a subset of C-fiber nociceptors in a model of painful diabetic neuropathy in the rat. Neuroscience 102, 185–192. Courteix, C., Eschalier, A., and Lavarenne, J. (1993). Streptozocin-induced diabetic rats: behavioural evidence for a model of chronic pain. PAIN 53, 81– 88. Daulhac, L., Mallet, C., Courteix, C., Etienne, M., Duroux, E., Privat, A. M., Eschalier, A., and Fialip, J. (2006). Diabetes-Induced Mechanical Hyperalgesia Involves Spinal Mitogen-Activated Protein Kinase Activation in Neurons and Microglia via N-Methyl-D-aspartate-Dependent Mechanisms. Molecular Pharmacology 70, 1246–1254. Dogrul, A., Ossipov, M. H., and Porreca, F. (2009). Differential mediation of descending pain facilitation and inhibition by spinal 5HT-3 and 5HT-7 receptors. Brain Research 1280, 52–59. 19 Fei, Z., Bera, T. K., Liu, X., Xiang, L., and Pastan, I. (2011). Ankrd26 gene disruption enhances adipogenesis of mouse embryonic fibroblasts. J Biol Chem 286, 27761–27768. Fukushima, T., Ohtsubo, T., Tsuda, M., Yanagawa, Y., and Hori, Y. (2009). Facilitatory Actions of Serotonin Type 3 Receptors on GABAergic Inhibitory Synaptic Transmission in the Spinal Superficial Dorsal Horn. Journal of Neurophysiology 102, 1459–1471. Galer, B. S., Gianas, A., and Jensen, M. P. (2000). Painful diabetic polyneuropathy: epidemiology, pain description, and quality of life. Diabetes Research and Clinical Practice 47, 123–128. Gao, Y.-J., and Ji, R.-R. (2009). c-Fos or pERK, Which is a Better Marker for Neuronal Activation and Central Sensitization After Noxious Stimulation and Tissue Injury? TOPAINJ 2, 11–17. Giesler, G. S., Gerhart, K. D., Yezierski, R. P., Wilcox, T. K., and Willis, W. D. (1981). Postsynaptic inhibition of primate spinothalamic neurons by stimulation in nucleus raphe magnus. Brain Research 204, 184–188. Glaum, S. R., Proudfit, H. K., and Anderson, E. G. (1990). 5-HT3 receptors modulate spinal nociceptive reflexes. Brain Research 510, 12–16. Guo, W., Miyoshi, K., Dubner, R., Gu, M., Li, M., Liu, J., Yang, J., Zou, S., Ren, K., Noguchi, K., et al. (2014). Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade. Molecular Pain 10, 35. Han, M., Huang, R.-Y., Du, Y.-M., Zhao, Z.-Q., and Zhang, Y.-Q. (2011). Early intervention of ERK activation in the spinal cord can block initiation of peripheral nerve injury-induced neuropathic pain in rats. Sheng Li Xue Bao 63, 106–114. Hwang, J. H., and Yaksh, T. L. (1997). The effect of spinal GABA receptor agonists on tactile allodynia in a surgically-induced neuropathic pain model in the rat. PAIN 70, 15–22. Ji, R. R., Baba, H., Brenner, G. J., and Woolf, C. J. (1999). Nociceptivespecific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat Neurosci 2, 1114–1119. Ji, R.-R., Befort, K., Brenner, G. J., and Woolf, C. J. (2002). ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. The Journal of Neuroscience 22, 478–485. 20 Jolivalt, C. G., Lee, C. A., Ramos, K. M., and Calcutt, N. A. (2008). Allodynia and hyperalgesia in diabetic rats are mediated by GABA and depletion of spinal potassium-chloride co-transporters. PAIN 140, 48–57. Kanazawa, A., Sawa, T., Akaike, T., and Maeda, H. (2002). Dietary lipid peroxidation products and DNA damage in colon carcinogenesis. Eur. J. Lipid Sci. Technol. 104, 439–447. Kaur, J., and Tikoo, K. (2013). p300/CBP dependent hyperacetylation of histone potentiates anticancer activity of gefitinib nanoparticles. Biochim Biophys Acta 1833, 1028–1040. Kawamata, T., Omote, K., Toriyabe, M., Yamamoto, H., and Namiki, A. (2003). The activation of 5-HT3 receptors evokes GABA release in the spinal cord. Brain Research 978, 1–6. Kia, H. K., Miquel, M. C., McKernan, R. M., Laporte, A. M., Lombard, M. C., Bourgoin, S., Hamon, M., and Verge, D. (1995). Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization. NeuroReport 6, 257–261. Kim, Y. S., Chu, Y., Han, L., Li, M., Li, Z., LaVinka, P. C., Sun, S., Tang, Z., Park, K., Caterina, M. J., et al. (2014). Central Terminal Sensitizationof TRPV1 by Descending Serotonergic Facilitation Modulates Chronic Pain. Neuron, 1–15. Lei, S., and Wilcox, G. L. (1990). Excitatory amino acid and opioid receptor regulation of spinal nociceptive neurotransmission. European Journal of Pharmacology 183, 1438–1439. Li, J.-Q., Chen, S.-R., Chen, H., Cai, Y.-Q., and Pan, H.-L. (2010). Regulation of increased glutamatergic input to spinal dorsal horn neurons by mGluR5 in diabetic neuropathic pain. J Neurochem 112, 162–172. McCleane, G. J., Suzuki, R., and Dickenson, A. H. (2003). Does a single intravenous injection of the 5HT3 receptor antagonist ondansetron have an analgesic effect in neuropathic pain? A double-blinded, placebo-controlled cross-over study. Anesth Analg 97, 1474–1478. Morgado, C., and Tavares, I. (2007). C-fos expression at the spinal dorsal horn of streptozotocin-induced diabetic rats. Diabetes Metab. Res. Rev. 23, 644–652. Morgado, C., Pereira-Terra, P., Cruz, C. D., and Tavares, I. (2011a). Minocycline completely reverses mechanical hyperalgesia in diabetic rats through microglia-induced changes in the expression of the potassium 27 Figure 3. 28 Table S1 - Blood glucose concentration, percentage of hemoglobin A1C and body weights of STZ-diabetic and control animals. Parameters STZ Control Blood Glucose concentration (mg/dl) 512.9 ± 10.15ª 121.3 ± 7.36 Hemoglobin A1C (%) 12.1 ± 0.25ª 4.8 ± 0.06 Body weight (g) 261.5 ± 6.55ª 402.1 ± 5.98 Independent sample t test. ªp < 0.0001. 29 Figure S1. 30 Figure S2. 31 ANEXOS 32 ANEXO 1 - Authors Guidelines (European Journal of Pain) Manuscript Structure and Word Count 1) Manuscript • Title page (see further details below) • Abstract (should not exceed 250 words, see further details below) • Text o Introduction (no subheadings, should not exceed 500 words) o Methods (or Literature Search Methods for Review Articles) o Results o Discussion and conclusions (should not exceed 1500 words) • Acknowledgements • Author contributions (see Section 6) • References (limited to 80 for original manuscripts) • Legends for illustrations and tables 2) Tables (to be uploaded as separate files) 3) Figures (to be uploaded as separate files) 4) Supporting material (additional material that will be published online-only, to be uploaded separately, see further details below) Title Page The title page should give: 1) The title of the article. Titles should be short and should not contain acronyms 2) A running head not exceeding 50 characters 3) The authors' names (initial(s) of first name(s) and last name of each author) 4) The names of the institutions at which the research was conducted, clearly linked to respective authors 5) The name, address, telephone and fax numbers, and e-mail address of the author responsible for correspondence 6) The category for which the manuscript is being submitted (original article, review, short communication) 7) A statement of all funding sources that supported the work 8) Any conflicts of interest disclosures (see Section 6). 9) Answers to each of the following questions in 2 or 3 bulleted statements (not exceeding 70 words): 'what's already known about this topic?' and 'what does this study add?'. For reviews only: 'database?' and ' what does this review add?'. Abstract The abstract should not exceed 250 words and should describe the background, the aims, the methods, the results and the conclusions reached. It should contain only 33 standard abbreviations and no references. For Original Manuscripts the following subheadings are required: • Background • Methods • Results • Conclusions For Reviews the following subheadings are required: • Background and Objective • Databases and Data Treatment • Results • Conclusions Acknowledgements The acknowledgements section should specify acknowledgement of technical help, but no sources of financial and material support. These should be given in the "Funding Sources" on the Title page. Author Contributions Authors are required to include a statement of responsibility at the end of their manuscript's text that specifies the contribution of every author (see Section 6). Please state that all authors discussed the results and commented on the manuscript. References If you use, e.g., Reference Manager, please note that EJP has adapted its reference style to the reference style of the journal Eur J Neuroscience. If you use, e.g., EndNote, please note that EJPuses the same reference style as the journal Neuron. In the text: references should be cited in parantheses at the appropriate point in the text by author(s) and year in chronological order, e.g., (Mustola, 1996; Baer, 1997; Mustola and Baer, 1998; Mustola et al., 1999). If two or more references with the same first author and year are cited, use lower-case letters a, b, etc., after the year both in the text and in the reference list. In the reference list: references to cited materials should be listed in alphabetical order at the end of the article. Please use Index Medicus abbreviations for journal titles. Include all authors. Do not use “et al.” in the reference list. Example for an article in a periodical: 34 De Peuter, S., Van Diest, I., Vansteenwegen, D. (2011). Understanding fear of pain in chronic pain: Interoceptive fear conditioning as a novel approach. Eur J Pain 15,889–894. Example for a chapter in a book: Janes, R., Saarto, T. (2010). Oncologic therapy in cancer pain. In Evidence-Based Chronic Pain Management, C. Stannard, E. Kalso, J. Ballantyne, eds. (Oxford: Wiley- Blackwell) pp. 311–326. Example for a book: Van Zundert, J., Patijn, J., Hartrick, C. (2011). Evidence-based Interventional Pain Practice (Oxford: Wiley-Blackwell). Citing and listing of Web references: As a minimum, the full URL should be given. Any further information, if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references should be listed separately (e.g., after the reference list) under the heading "Web references". Personal communications, manuscripts in preparation and other unpublished data should not be cited in the reference list but may be mentioned in the text in parentheses. Figures All colour illustrations will be published free of charge. All figures must be uploaded as separate files. Figure legends should be listed on a separate pagein numerical order and should contain brief but comprehensible explanations. Figures should be referred to in the text in numerical sequence as follows: Fig. 1, Figs 2–4. The place at which a figure is to be inserted in the printed text should be indicated clearly on a manuscript. Where a figure has more than one panel, each panel should be labeled in the top left-hand corner using lower case letters in parentheses i.e. ‘(a)’, ‘(b)’ etc., and a brief description of each panel given in the figure legend. Authors are themselves responsible for obtaining permission to reproduce previously published figures or tables. When an individual is identifiable in a photograph written consent must be obtained. This permission must include the right to publish in electronic media. Print publication requires high quality, EPS (lineart) or TIFF/PDF (halftone/photographs) files are preferable (though GIF, JPEG, PICT or Bitmap files are acceptable for submission). MS PowerPoint and Word Graphics are unsuitable for printed pictures. Scans (TIFF only) should have a resolution of 300 dpi (halftone) or 600 to 1200 dpi (line drawings) in relation to the reproduction size (see below). EPS files should be saved with fonts embedded (and with a TIFF preview if possible). For scanned images, the scanning resolution (at final image size) should 35 be as follows to ensure good reproduction: lineart: >600 dpi; half-tones: >300 dpi; figures containing both halftone and line images: >600 dpi. Detailed instructions for electronic artwork preparation may be found at http://authorservices.wiley.com/bauthor/illustration.asp. Tables Tables should be referred to in the text in numerical sequence as follows: Table 1, Table 2. Each table, with an appropriate brief legend, comprehensible without reference to the text, should be typed on a separate page. For footnotes, use superscripts 'a', 'b', 'c', etc., not asterisks or other symbols. Supplementary Materials to be published online-only EJP encourages the submission of underlying data sets, appendices, additional figures or tables, movie files, animations, etc. as online-only supporting information. Supporting information should be uploaded during manuscript submission (see Section 3). Supporting information should be important ancillary information that is relevant to the parent article but which does not or cannot appear in the printed edition of the journal. To submit any material to be published online only please do the following: - choose the item "table" or "figure" as usual when uploading the files of tables or figures - choose the item "manuscript" if you want to publish certain parts of the manuscript online only and upload the related word file - scroll down the page and enter in the "description"-box the kind of material you want to submit online only. Please use the following terms: - for tables: "tableS1", "tableS2" etc. - for figures: "figureS1, "figureS2" etc. The figure legends should be included in the figure's file. - for parts of the manuscript's text: "methodsS1", resultsS1", "discussionS1" or "AppendixS1" (please note that it is not possible to publish additional material for the introduction) - please indicate and cite clearly in your manuscript the online only material using the terms given above. Units & Abbreviations Measurements of length, height and volume should be reported in metric units (metre, kilogram, litre). Temperatures should be given in degrees Celsius and blood pressures in millimetres of mercury or kPa with the alternative units in parentheses. All other measurements including laboratory measurements should be reported in the metric system in terms of the International System of Units (SI). 36 Abbreviations should be limited and defined after the first use of the term. Drug Names Generic names of drugs should be used where possible. When quoting from specific materials on proprietary drugs, authors must state in parentheses the name and address of the manufacturer.