Glial Inhibitor Research Articles

Minocycline and fluorocitrate suppress spinal nociceptive signaling in intrathecal IL‐1β–induced thermal hyperalgesic rats

We previously demonstrated that intrathecal IL-1β caused thermal hyperalgesia in rats. This study was conducted to examine the effects and cellular mechanisms of glial inhibitors on IL-1β-induced nociception in rats. The effects of minocycline (20 μg), fluorocitrate (1 nmol), and SB203580 (5 μg) on IL-1β (100 ng) treatment in rats were measured by nociceptive behaviors, western blotting of p38 mitogen-activated protein kinase (MAPK) and inducible nitric oxide synthase (iNOS) expression, cerebrospinal fluid nitric oxide (NO) levels, and immunohistochemical analyses. The results demonstrated that intrathecal IL-1β activated microglia and astrocytes, but not neurons, in the dorsal horn of the lumbar spinal cord, as evidenced by morphological changes and increased immunoreactivity, phosphorylated p38 (P-p38) MAPK, and iNOS expression; the activation of microglia and astrocytes peaked at 30 min and lasted for 6 h. The immunoreactivities of microglia and astrocytes were significantly increased at 30 min (6.6- and 2.7-fold, respectively) and 6 h (3.3- and 4.0-fold, respectively) following IL-1β injection, as compared with saline controls at 30 min (all P < 0.01). IL-1β induced P-p38 MAPK and iNOS expression predominantly in microglia and less in astrocytes. Minocycline, fluorocitrate, or SB203580 pretreatment suppressed this IL-1β-upregulated P-p38 MAPK mainly in microglia and iNOS mainly in astrocytes; minocycline exhibited the most potent effect. Minocycline and fluorocitrate pretreatment abrogated IL-1β-induced NO release and thermal hyperalgesia in rats. In conclusion, minocycline, fluorocitrate, and SB203580 effectively suppressed the IL-1β-induced central sensitization and hyperalgesia in rats.

Combination of Tramadol with Minocycline Exerted Synergistic Effects on a Rat Model of Nerve Injury-Induced Neuropathic Pain

Neuropathic pain is a refractory clinical problem. Certain drugs, such as tramadol, proved useful for the treatment of neuropathic pain by inhibiting the activity of nociceptive neurons. Moreover, studies indicated that suppression or modulation of glial activation could prevent or reverse neuropathic pain, for example with the microglia inhibitor minocycline. However, few present clinical therapeutics focused on both neuronal and glial participation when treating neuropathic pain. Therefore, the present study hypothesized that combination of tramadol with minocycline as neuronal and glial activation inhibitor may exert some synergistic effects on spinal nerve ligation (SNL)-induced neuropathic pain. Intrathecal tramadol or minocycline relieved SNL-induced mechanical allodynia in a dose-dependent manner. SNL-induced spinal dorsal horn Fos or OX42 expression was downregulated by intrathecal tramadol or minocycline. Combination of tramadol with minocycline exerted powerful and synergistic effects on SNL-induced neuropathic pain also in a dose-dependent manner. Moreover, the drug combination enhanced the suppression effects on SNL-induced spinal dorsal horn Fos and OX42 expression, compared to either drug administered alone. These results indicated that combination of tramadol with minocycline could exert synergistic effects on peripheral nerve injury-induced neuropathic pain; thus, a new strategy for treating neuropathic pain by breaking the interaction between neurons and glia bilaterally was also proposed.

Axonal regeneration induced by blockade of glial inhibitors coupled with activation of intrinsic neuronal growth pathways

Several pharmacological approaches to promote neural repair and recovery after CNS injury have been identified. Blockade of either astrocyte-derived chondroitin sulfate proteoglycans (CSPGs) or oligodendrocyte-derived NogoReceptor (NgR1) ligands reduces extrinsic inhibition of axonal growth, though combined blockade of these distinct pathways has not been tested. The intrinsic growth potential of adult mammalian neurons can be promoted by several pathways, including pre-conditioning injury for dorsal root ganglion (DRG) neurons and macrophage activation for retinal ganglion cells (RGCs). Singly, pharmacological interventions have restricted efficacy without foreign cells, mechanical scaffolds or viral gene therapy. Here, we examined combinations of pharmacological approaches and assessed the degree of axonal regeneration. After mouse optic nerve crush injury, NgR1−/− neurons regenerate RGC axons as extensively as do zymosan-injected, macrophage-activated WT mice. Synergistic enhancement of regeneration is achieved by combining these interventions in zymosan-injected NgR1−/− mice. In rats with a spinal dorsal column crush injury, a preconditioning peripheral sciatic nerve axotomy, or NgR1(310)ecto-Fc decoy protein treatment or ChondroitinaseABC (ChABC) treatment independently support similar degrees of regeneration by ascending primary afferent fibers into the vicinity of the injury site. Treatment with two of these three interventions does not significantly enhance the degree of axonal regeneration. In contrast, triple therapy combining NgR1 decoy, ChABC and preconditioning, allows axons to regenerate millimeters past the spinal cord injury site. The benefit of a pre-conditioning injury is most robust, but a peripheral nerve injury coincident with, or 3days after, spinal cord injury also synergizes with NgR1 decoy and ChABC. Thus, maximal axonal regeneration and neural repair are achieved by combining independently effective pharmacological approaches.

Acute Neuroimmune Modulation Attenuates the Development of Anxiety-Like Freezing Behavior in an Animal Model of Traumatic Brain Injury

Chronic anxiety is a common and debilitating result of traumatic brain injury (TBI) in humans. While little is known about the neural mechanisms of this disorder, inflammation resulting from activation of the brain's immune response to insult has been implicated in both human post-traumatic anxiety and in recently developed animal models. In this study, we used a lateral fluid percussion injury (LFPI) model of TBI in the rat and examined freezing behavior as a measure of post-traumatic anxiety. We found that LFPI produced anxiety-like freezing behavior accompanied by increased reactive gliosis (reflecting neuroimmune inflammatory responses) in key brain structures associated with anxiety: the amygdala, insula, and hippocampus. Acute peri-injury administration of ibudilast (MN166), a glial cell activation inhibitor, suppressed both reactive gliosis and freezing behavior, and continued neuroprotective effects were apparent several months post-injury. These results support the conclusion that inflammation produced by neuroimmune responses to TBI play a role in post-traumatic anxiety, and that acute suppression of injury-induced glial cell activation may have promise for the prevention of post-traumatic anxiety in humans.

Leptin action on nonneuronal cells in the CNS: potential clinical applications

Leptin, an adipocyte-derived cytokine, crosses the blood–brain barrier to act on many regions of the central nervous system (CNS). It participates in the regulation of energy balance, inflammatory processes, immune regulation, synaptic formation, memory condensation, and neurotrophic activities. This review focuses on the newly identified actions of leptin on astrocytes. We first summarize the distribution of leptin receptors in the brain, with a focus on the hypothalamus, where the leptin receptor is known to mediate essential feeding suppression activities, and on the hippocampus, where leptin facilitates memory, reduces neurodegeneration, and plays a dual role in seizures. We will then discuss regulation of the nonneuronal leptin system in obesity. Its relationship with neuronal leptin signaling is illustrated by in vitro assays in primary astrocyte culture and by in vivo studies on mice after pretreatment with a glial metabolic inhibitor or after cell-specific deletion of intracellular signaling leptin receptors. Overall, the glial leptin system shows robust regulation and plays an essential role in obesity. Strategies to manipulate this nonneuronal leptin signaling may have major clinical impact.

179 Enteric Glia Mediate Nicotinic-Stimulated Epithelial Ion Transport in Mouse Colon Revealed by Acute Glial Metabolic Inhibition

179 Enteric Glia Mediate Nicotinic-Stimulated Epithelial Ion Transport in Mouse Colon Revealed by Acute Glial Metabolic Inhibition

Inhibition of glial activation in rostral ventromedial medulla attenuates mechanical allodynia in a rat model of cancer-induced bone pain

Descending nociceptive modulation from the supraspinal structures plays an important role in cancer-induced bone pain (CIBP). Rostral ventromedial medulla (RVM) is a critical component of descending nociceptive facilitation circuitry, but so far the mechanisms are poorly known. In this study, we investigated the role of RVM glial activation in the descending nociceptive facilitation circuitry in a CIBP rat model. CIBP rats showed significant activation of microglia and astrocytes, and also up-regulation of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and pro-inflammatory mediators released by glial cells (IL-1β, IL-6, TNF-α and brain-derived neurotrophic factor) in the RVM. Stereotaxic microinjection of the glial inhibitors (minocycline and fluorocitrate) into CIBP rats' RVM could reverse the glial activation and significantly attenuate mechanical allodynia in a time-dependent manner. RVM microinjection of p38 MAPK inhibitor (SB203580) abolished the activation of microglia, reversed the associated up-regulation of pro-inflammatory mediators and significantly attenuated mechanical allodynia. Taken together, these results suggest that RVM glial activation is involved in the pathogenesis of CIBP. RVM microglial p38 MAPK signaling pathway is activated and leads to the release of downstream pro-inflammatory mediators, which contribute to the descending facilitation of CIBP.

Evidence that glial cells modulate synaptic transmission in the NTS neurons sending projections to RVLM

The peripheral chemoreflex activation produces autonomic and respiratory responses. In previous study we verified that ATP modulates the glutamatergic transmission onto NTS neurons sending projections to RVLM, which are related to the sympathoexcitatory component of chemoreflex. Here, we evaluated the role of glial cells in ATP release and the purinergic subtype receptors involved with this modulation. Adult rats received fluorescent tracer microinjections into RVLM and 7 days later the labeled NTS neurons sending projections to RVLM were identified in brainstem slices. TS evoked excitatory pos‐synaptic currents (TS‐eEPSCs) were recorded using patch clamp. Inhibition of glial cells with fluoracetate (FAC− 1mM) decreased the amplitude of TS‐eEPSCs and this effect was independent of glutamine in the bath (FAC: − 56 ± 0.06%, n=6; FAC + glutamine: −65 ± 0.11%, n=10). iso‐PPADS (100 μM), a P2X receptor antagonist, or A317491 (10 μM), a P2X3 or P2X2/3 receptor antagonist, produced similar decrease in the amplitude of TS‐eEPSCs (iso‐PPADS: −58 ± 7%, n=6; A317491: −57± 5%, n=5%). The subsequent addition of iso‐PPADS in the bath during FAC perfusion did not alter the glial inhibition effect on TS‐eEPSCs (FAC: −58 ± 11%; iso‐PPADS + FAC: −45 ± 6%, n=5). We conclude that glial cells modulate the synaptic transmission in the NTS neurons sending projections to RVLM. Supported by FAPESP and CNPq.

Distinct time courses of microglial and astrocytic hyperactivation and the glial contribution to pain hypersensitivity in a facial cancer model

Although recent evidence suggests that central glial hyperactivation is involved in cancer-induced persistent pain, the time course of this hyperactivation and the glial contribution to pain hypersensitivity remain unclear. The present study investigated the time-dependent spatial changes of microglial and astrocytic hyperactivation in the trigeminocervical complex, which consists of the medullary (MDH) and upper cervical (UCDH) dorsal horns, and pain-related behaviors in a rat facial cancer model in which Walker 256B-cells are inoculated into the vibrissal pad. In this model, the tumors grew within the vibrissal pad, from which sensory nerve fibers project into the MDH, but did not expand into the infraorbital region, from which fibers project into the UCDH. Nevertheless, mechanical allodynia and thermal hyperalgesia were observed not only in the vibrissal pad but also in the infraorbital region. Western blotting and immunofluorescence studies indicated that microglia were widely activated in the trigeminocervical complex on day 4 and gradually inactivated by day 11. In contrast, astrocytes were only activated in the MDH on day 4; the hyperactivation later expanded into the UCDH. Daily administration of the glial hyperactivation inhibitor propentofylline beginning on day 4 suppressed the glial hyperactivation on later days. Propentofylline treatment largely prevented allodynia/hyperalgesia in the infraorbital region beginning on day 5, although established allodynia/hyperalgesia in the vibrissal pad was less sensitive to the treatment. These results suggest that central glial hyperactivation, transient microglial hyperactivation and persistent astrocytic hyperactivation, contributes to the development of pain hypersensitivity but not to the maintenance of pain in this model.

Contribution of Microglia and Astrocytes to the Central Sensitization, Inflammatory and Neuropathic Pain in the Juvenile Rat

BackgroundThe development of pain after peripheral nerve and tissue injury involves not only neuronal pathways but also immune cells and glia. Central sensitization is thought to be a mechanism for such persistent pain, and ATP involves in the process. We examined the contribution of glia to neuronal excitation in the juvenile rat spinal dorsal horn which is subjected to neuropathic and inflammatory pain.ResultsIn rats subjected to neuropathic pain, immunoreactivity for the microglial marker OX42 was markedly increased. In contrast, in rats subjected to inflammatory pain, immunoreactivity for the astrocyte marker glial fibrillary acidic protein was increased slightly. Optically-recorded neuronal excitation induced by single-pulse stimulation to the dorsal root was augmented in rats subjected to neuropathic and inflammatory pain compared to control rats. The bath application of a glial inhibitor minocycline and a p38 mitogen-activated protein kinase inhibitor SB203580 inhibited the neuronal excitation in rats subjected to neuropathic pain. A specific P2X1,2,3,4 antagonist TNP-ATP largely inhibited the neuronal excitation only in rats subjected to neuropathic pain rats. In contrast, an astroglial toxin L-alpha-aminoadipate, a gap junction blocker carbenoxolone and c-Jun N-terminal kinase inhibitor SP600125 inhibited the neuronal excitation only in rats subjected to inflammatory pain. A greater number of cells in spinal cord slices from rats subjected to neuropathic pain showed Ca2+ signaling in response to puff application of ATP. This Ca2+ signaling was inhibited by minocycline and TNP-ATP.ConclusionsThese results directly support the notion that microglia is more involved in neuropathic pain and astrocyte in inflammatory pain.

Antinociceptive interaction of (±)-CPP and propentofylline in monoarthritic rats

IntroductionMultiple studies have shown that glial cells of the spinal cord, such as astrocytes and microglia, have close contact with neurons, suggesting the term tripartite synapse. In these synapses, astrocytes surrounding neurons contribute to neuronal excitability and synaptic transmission, thereby increasing nociception and thus the persistence of chronic pain. Conversely, the N-methyl-D-aspartate (NMDA) receptor is crucial in the generation and maintenance of chronic pain. It has multiple sites of modulation. One is the site of recognition of extracellular neurotransmitter (glutamate), which can be blocked by competitive antagonists such as (3-(2-carboxipiperazin-4)1-propyl phosphonic acid), (±)-CPP, resulting in a blockade of the calcium current and thus the intracellular transduction process. In the present study, we investigated whether the potential antinociceptive effect of glial inhibition produced by propentofylline (PPF) can be enhanced when combined with an NMDA-receptor inhibitor such as (±)-CPP.MethodsWe used Sprague-Dawley monoarthritic rats. The monoarthritis was induced by injection of complete Freund adjuvant in the right tibiotarsal joint. Four weeks later, rats were treated with PPF (1, 10, 30, and 100 μg/10 μl) intrathecally (i.t.) for 10 days, injected once with (±)-CPP (2.5, 5, 12.5, 25, 50, and 100 μg/10 μl, i.t.), or both treatments combined. The antinociceptive effect was evaluated on day 11 for PPF and immediately to (±)-CPP, by assessing the vocalization threshold to mechanical stimulation of the arthritic paw.ResultsThe data indicate that intrathecal administration of increasing concentrations of (±)-CPP or PPF produced a significant dose-dependent antinociceptive effect with respect to monoarthritic rats receiving saline. The linear regression analysis showed that the dose that produces 30% of maximal effect (ED30) for i.t. (±)-CPP was 3.97 μg, and 1.42 μg for i.t. PPF. The administration of the PPF and (±)-CPP combination in fixed proportions of ED30 produced a dose-dependent antinociceptive effect, showing an interaction of the supraadditive type.ConclusionsThe results suggest that glia inhibitors can synergically potentiate the effect of glutamate blockers for the treatment of chronic inflammatory pain.

Activation of satellite glial cells in lumbar dorsal root ganglia contributes to neuropathic pain after spinal nerve ligation

The role of satellite glial cells (SGCs) of sensory ganglia in chronic pain begins to receive interest. The present study aims to investigate the contribution of SGC activation to the development of neuropathic pain. A neuropathic pain model was established by lumbar 5 spinal nerve ligation (SNL), and glial fibrillary acidic protein (GFAP) was used as a marker of SGC activation. It was found that SGCs were activated in the ipsilateral dorsal root ganglia (DRG) increased significantly as early as 4 h following SNL, gradually increased to a peak level at day 7, and then stayed at a high level to the end of the experiment at day 56. SGC activation in the SNL group was significantly higher than that in the sham group at days 1, 3 and 7 after operation. Immunofluorescent double labeling showed that the activated SGCs encircled large, medium-sized and small neurons. The SGCs surrounded the small and medium-sized neurons were preferentially activated in the early phase, but shifted to large diameter neurons as time went on. Continuous infusion of fluorocitrate, a glial metabolism inhibitor, to the affected DRG via mini-osmotic pump for 7 d significantly alleviated mechanical allodynia at day 7. These results suggest that SGCs in the DRG were activated after SNL. SGC activation contributed to the early maintenance of neuropathic pain.

Controlled Adhesion and Growth of Long Term Glial and Neuronal Cultures on Parylene-C

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO2 substrate. We harvested glia and neurons from the Sprague-Dawley (P1–P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures.

The effect of intrathecal administration of glial activation inhibitors on dorsal horn BDNF overexpression and hind paw mechanical allodynia in spinal nerve ligated rats

Recent studies have suggested that activated glia in the spinal cord may play a vital role at different times during spinal nerve ligation (SNL)-induced neuropathic pain; therefore, glial activation inhibitors have been used as effective painkillers. Brain-derived neurotrophic factor (BDNF) is also known to be a powerful pain modulator, but it remains unclear how it contributes to the glial activation inhibitor-based treatment. This study revealed the following results: (1) intrathecal administration of minocycline (a microglial activation inhibitor) could prevent mechanical allodynia during the initiation of SNL-induced neuropathic pain, and its action was associated with the elimination of BDNF overexpression in the dorsal horn; (2) the spinal injection of fluorocitrate (an astrocytic activation inhibitor) but not minocycline could reverse mechanical allodynia during the maintenance phase of SNL-induced pain, and its action was also related to a decrease in BDNF overexpression in the dorsal horn; and (3) treatment with TrkB/Fc (a BDNF-sequestering protein) had a similar effect during both the early development and maintenance periods. These results led to the following conclusions: (1) elevated BDNF expression in the dorsal horn was required to develop and maintain neuropathic pain; (2) minocycline could only prevent mechanical allodynia in the early stages, possibly by inhibiting BDNF release from microglia; and (3) fluorocitrate could reverse existing mechanical allodynia, and its action was associated with the inhibition of BDNF upregulation induced by astrocytic activation.

Chemical stimulation of the ST36 acupoint reduces both formalin-induced nociceptive behaviors and spinal astrocyte activation via spinal alpha-2 adrenoceptors

Spinal astrocytes have emerged as important mechanistic contributors to pathological and chronic pain. Recently, we have demonstrated that injection of diluted bee venom (DBV) into the Zusanli (ST36) acupoint produces a potent anti-nociceptive effect via the activation of spinal alpha-2 adrenoceptors. However, it is unclear if this anti-nociceptive effect is associated with alterations in spinal astrocytes. Thus, the present study was designed to determine: (1) whether DBV's anti-nociceptive effect in the formalin test involves suppression of spinal astrocyte activation; (2) whether DBV-induced astrocyte inhibition is mediated by spinal alpha-2 adrenoceptors; and (3) whether this glial modulation is potentiated by intrathecal administration of the glial metabolic inhibitor, fluorocitrate (FC) in combination with DBV injection. DBV was injected directly into the ST36 acupoint, and spinal expression of the astrocytic marker, glial fibrillary acidic protein (GFAP), was assessed together with effects on formalin-induced nociception. DBV treatment reduced pain responses in the late phase of the formalin test and significantly blocked the formalin-evoked increase in spinal GFAP expression. These effects of DBV were prevented by intrathecal pretreatment with selective alpha-2A and alpha-2C adrenoceptor antagonists. Moreover, low dose intrathecal injection of FC in conjunction with low dose DBV injection into the ST36 acupoint synergistically suppressed pain responses and GFAP expression. These results demonstrate that DBV stimulation of the ST36 acupoint inhibits the formalin-induced activation of spinal astrocytes and nociceptive behaviors in this inflammatory pain model and this inhibition is associated with the activation of spinal alpha-2 adrenoceptors.

115. Diagnostic value of cytokine profiling in chronic pain disorders and as a biomarker of induced pain after capsaicin

Glia plays a crucial role in the maintenance of neuronal homeostasis in the central nervous system. The microglial production of immune factors is believed to play an important role in nociceptive transmission. Pain may now be considered a neuro-immune disorder, since it is known that the activation of immune and immune-like glial cells in the dorsal root ganglia and spinal cord results in the release of both pro- and anti-inflammatory cytokines, as well as algesic and analgesic mediators. In this review we presented an important role of cytokines (IL-1alfa, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-15, IL-18, TNFalpha, IFNgamma, TGF-beta 1, fractalkine and CCL2); complement components (C1q, C3, C5); metaloproteinases (MMP-2,-9) and many other factors, which become activated on spinal cord and DRG level under neuropathic pain. We discussed the role of the immune system in modulating chronic pain. At present, unsatisfactory treatment of neuropathic pain will seek alternative targets for new drugs and it is possible that anti-inflammatory factors like IL-10, IL-4, IL-1alpha, TGF-beta 1 would fulfill this role. Another novel approach for controlling neuropathic pain can be pharmacological attenuation of glial and immune cell activation. It has been found that propentofylline, pentoxifylline, minocycline and fluorocitrate suppress the development of neuropathic pain. The other way of pain control can be the decrease of pro-nociceptive agents like transcription factor synthesis (NF-kappaB, AP-1); kinase synthesis (MEK, p38MAPK, JNK) and protease activation (cathepsin S, MMP9, MMP2). Additionally, since it is known that the opioid-induced glial activation opposes opioid analgesia, some glial inhibitors, which are safe and clinically well tolerated, are proposed as potential useful ko-analgesic agents for opioid treatment of neuropathic pain. This review pointed to some important mechanisms underlying the development of neuropathic pain, which led to identify some possible new approaches to the treatment of neuropathic pain, based on the more comprehensive knowledge of the interaction between the nervous system and glial and immune cells.

Do glial cells in the NTS play a role in autonomic and respiratory control in rats?

Background/Aims: In the last two decades, glial cells have been implicated in a variety of neurophysiological processes, including synaptic regulation, neural network synchronization, respiratory rhythm generation and central chemoreception. In the present study, we investigated whether glial inhibition in the NTS, an important nucleus for the processing of visceral input and respiratory pattern control, could affect baseline respiratory and autonomic regulation in rats.

TNFα is involved in neuropathic pain induced by nucleoside reverse transcriptase inhibitor in rats

In patients with HIV/AIDS, neuropathic pain is a common neurological complication. Infection with the HIV itself may lead to neuropathic pain, and painful symptoms are enhanced when patients are treated with nucleoside reverse transcriptase inhibitors (NRTIs). The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current studies, we tested the role of TNFα in antiretroviral drug-induced neuropathic pain. We administered 2',3'-dideoxycytidine (ddC, one of the NRTIs) systemically to induce mechanical allodynia. We found that ddC induced overexpression of both mRNA and proteins of GFAP and TNFα in the spinal dorsal horn. TNFα was colocalized with GFAP in the spinal dorsal horn and with NeuN in the DRG. Knockdown of TNFα with siRNA blocked the mechanical allodynia induced by ddC. Intrathecal administration of glial inhibitor or recombinant TNF soluble receptor, reversed mechanical allodynia induced by ddC. These results suggest that TNFα is involved in NRTI-induced neuropathic pain.

Up-regulation of NOS expression, activity and no production in the spinal cord induced by acute myositis in rats

Glial cells have an important role in inflammatory processes, through the release of several mediators. In order to explore the involvement of glia in the nitric oxide(NO)-mediated nociception, the present study was undertaken to investigate the NO synthase (NOS) expression in the spinal cord during the process of acute myositis induced by carrageenan in rats. In addition, we will also evaluate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression. Myositis was induced by the administration of 400μg of carrageenan (Cg) in the right gastrocnemius (GS) muscle of rats, with saline (150μL) as control. Immunohistochemical assays to evaluate nNOS expression were performed in the lumbar dorsal horn and central canal of spinal cord 2h after the myositis-induced. The activity of all NOS isoforms was detected by NADPH-d histochemistry staining. Intrathecal injection of fluorocitrate was used to examine potential spinal glial mediation in this model. The immunohistochemical data showed that carrageenan up-regulated nNOS expression 2h after acute myositis, as immunostaining to nNOS was significantly higher in animals injected with Cg when compared with controls groups. Fluorocitrate, a glial inhibitor, when administered by intrathecal route, was not able to decrease nNOS expression in rats with myositis. Regarding NADPH-d, we also observed and increase of NO positive cells after carrageenan injections when compared with control animals. Our results suggest that in the process of acute muscle inflammation occurs the release of NO, through the activation of positive NOS neurons in the dorsal horn and central canal of the spinal cord. Understanding the role of glial cells during myositis-induced nociception may lead to new strategies for management of muscle pain, one of the most frequent types of clinically relevant pain. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Glial cells have an important role in inflammatory processes, through the release of several mediators. In order to explore the involvement of glia in the nitric oxide(NO)-mediated nociception, the present study was undertaken to investigate the NO synthase (NOS) expression in the spinal cord during the process of acute myositis induced by carrageenan in rats. In addition, we will also evaluate the effect of fluorocitrate (FC), an inhibitor of glial metabolism, on NOS expression. Myositis was induced by the administration of 400μg of carrageenan (Cg) in the right gastrocnemius (GS) muscle of rats, with saline (150μL) as control. Immunohistochemical assays to evaluate nNOS expression were performed in the lumbar dorsal horn and central canal of spinal cord 2h after the myositis-induced. The activity of all NOS isoforms was detected by NADPH-d histochemistry staining. Intrathecal injection of fluorocitrate was used to examine potential spinal glial mediation in this model. The immunohistochemical data showed that carrageenan up-regulated nNOS expression 2h after acute myositis, as immunostaining to nNOS was significantly higher in animals injected with Cg when compared with controls groups. Fluorocitrate, a glial inhibitor, when administered by intrathecal route, was not able to decrease nNOS expression in rats with myositis. Regarding NADPH-d, we also observed and increase of NO positive cells after carrageenan injections when compared with control animals. Our results suggest that in the process of acute muscle inflammation occurs the release of NO, through the activation of positive NOS neurons in the dorsal horn and central canal of the spinal cord. Understanding the role of glial cells during myositis-induced nociception may lead to new strategies for management of muscle pain, one of the most frequent types of clinically relevant pain. Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

Remodelling of the sympathetic nervous system following peripheral inflammation

Remodelling of the sympathetic innervation of the colon occurs during inflammatory bowel disease (IBD). However, the mechanisms are unclear. We hypothesized that interleukin (IL)-17A, a key cytokine in IBD pathogenesis, plays a role in this remodelling. Chronic DSS-induced colitis in mice and immunohistochemistry were used to study sympathetic neuroplasticity. Sympathetic neurite outgrowth was examined from superior mesenteric ganglia (SMG) neurons cultured in the presence of IL-17A. Electrophysiology and Ca2+ imaging techniques were also used. Colitis enhanced sympathetic innervation of the colonic myenteric plexus and mucosa. Glial inhibition and explant studies suggest that IL-17A acts directly on axons to promote SMG outgrowth. Incubation of SMG neurons in IL-17A reduced voltage-gated Ca2+ current (ICa) and depolarisation-induced Ca2+ influx in growth cones. This effect was abrogated by an inhibitor of NF-κB signalling which also prevented IL-17A-induced neurite outgrowth. Blockade of ICa with a VGC channel blocker reproduced the effects of IL-17A on axonal outgrowth. Therefore, IL-17A may contribute to sympathetic remodelling during IBD through activation of NF-κB and inhibition of Ca2+ influx.