TLR4 Antagonism Reduces Movement-Induced Nociception and ATF-3 Expression in Experimental Osteoarthritis.

INTRODUCTION: Toll-like receptor 4 (TLR4) is a pattern recognition receptor involved in the detection of pathogen-associated molecular patterns (PAMPs), but also a "danger-sensing" receptor that recognizes host-derived endogenous molecules called damage-associated molecular patterns (DAMPs). The involvement of TLR4 in rheumatic diseases is becoming evident, as well as its potential role as a target for therapeutic intervention. Moreover, increasing evidence also suggests that TLR4 is implicated in chronic pain states. Thus, in this study, we evaluated whether a systemic administration of a synthetic antagonist of TLR4 (TLR4-A1) could decrease nociception and cartilage degradation in experimental osteoarthritis (OA). Furthermore, as the activation transcription factor (ATF)-3 serves as a negative regulator for TLR4-stimulated inflammatory response, we also evaluated the effect of TLR4 inhibition on ATF-3 expression in primary afferent neurons at the dorsal root ganglia (DRG). METHODS: OA was induced in adult male Wistar rats through an intra-articular injection of 2 mg of sodium mono-iodoacetate (MIA) into the left knee. From days 14 to 28 after OA induction, animals received an intraperitoneal injection of either TLR4-A1 (10 mg/kg) or vehicle. Movement- and loading-induced nociception was evaluated in all animals, by the Knee-Bend and CatWalk tests, before and at several time-points after TLR4-A1/vehicle administration. Immunofluorescence for TLR4 and ATF-3 was performed in L3-L5 DRG. Knee joints were processed for histopathological evaluation. RESULTS: Administration of TLR4-A1 markedly reduced movement-induced nociception in OA animals, particularly in the Knee-Bend test. Moreover, the increase of ATF-3 expression observed in DRG of OA animals was significantly reduced by TLR4-A1. However, no effect was observed in cartilage loss nor in the neuronal cytoplasmic expression of TLR4 upon antagonist administration. CONCLUSION: The TLR4 antagonist administration possibly interrupts the TLR4 signalling cascade, thus decreasing the neurotoxic environment at the joint, which leads to a reduction in ATF-3 expression and in nociception associated with experimental OA.

Amylin, a peptide expressed by nociceptors, modulates chronic neuropathic pain.

BACKGROUND: Amylin is a calcitonin gene-related peptide family member expressed by nociceptors. Amylin's expression is down-regulated following nerve damage, and studies suggested it affects nociception. We aimed at clarifying amylin's effects on chronic neuropathic pain and investigating its site of action. METHODS: Chronic neuropathic pain was induced in rats by spared nerve injury (SNI) surgery. Mechanical allodynia/hyperalgesia and cold allodynia/hyperalgesia were assessed by the von Frey, pinprick, acetone and cold plate behavioural tests, respectively. Amylin, amylin-receptor antagonist (AC187) or vehicle solutions were delivered chronically, by a subcutaneous (SC) mini-osmotic pump, or acutely, by SC or intrathecal (IT) injections. Cellular and fibre markers were used to detect spinal cord alterations in SNI rats after chronic amylin administration. RESULTS: Continuous subcutaneous amylin administration aggravated cold allodynia in SNI animals, possibly via amylin-receptors (AmyR) in supraspinal areas. Acute intrathecal administration of amylin attenuated mechanical hyperalgesia, whereas AC187 reduced mechanical allodynia, suggesting distinct roles of endogenous amylin and of pharmacological amylin doses when targeting spinal cord amylin receptors. Chronic amylin administration promoted c-Fos activation only in the dorsal horn neurons of SHAM animals, suggesting a distinctive role of amylin in the activation of the spinal neuronal circuitry under neuropathic and physiological conditions. ERK1/2 phosphorylation increased in the dorsal horn neurons of SNI rats chronically treated with amylin. This ERK1/2 cascade activation may be related to amylin's effect on the aggravation of cold allodynia in SNI rats. CONCLUSIONS: Amylin's nociceptive effects seem to depend on the treatment duration and route of administration by acting at different levels of the nervous system. SIGNIFICANCE: Amylin modulated neuropathic pain by acting at different levels of the nervous system. Whereas supraspinal areas may be involved in amylin's induced pronociception, modulation of spinal cord amylin receptors by endogenous or pharmacological amylin doses triggers both pro- and antinociceptive effects.

Drug-Induced HSP90 Inhibition Alleviates Pain in Monoarthritic Rats and Alters the Expression of New Putative Pain Players at the DRG.

Purinergic receptors (P2XRs) have been widely associated with pain states mostly due to their involvement in neuron-glia communication. Interestingly, we have previously shown that satellite glial cells (SGC), surrounding dorsal root ganglia (DRG) neurons, become activated and proliferate during monoarthritis (MA) in the rat. Here, we demonstrate that P2X7R expression increases in ipsilateral DRG after 1 week of disease, while P2X3R immunoreactivity decreases. We have also reported a significant induction of the activating transcriptional factor 3 (ATF3) in MA. In this study, we show that ATF3 knocked down in DRG cell cultures does not affect the expression of P2X7R, P2X3R, or glial fibrillary acidic protein (GFAP). We suggest that P2X7R negatively regulates P2X3R, which, however, is unlikely mediated by ATF3. Interestingly, we found that ATF3 knockdown in vitro induced significant decreases in the heat shock protein 90 (HSP90) expression. Thus, we evaluated in vivo the involvement of HSP90 in MA and demonstrated that the HSP90 messenger RNA levels increase in ipsilateral DRG of inflamed animals. We also show that HSP90 is mostly found in a cleaved form in this condition. Moreover, administration of a HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), attenuated MA-induced mechanical allodynia in the first hours. The drug also reversed the HSP90 upregulation and cleavage. 17-DMAG seemed to attenuate glial activation and neuronal sensitization (as inferred by downregulation of GFAP and P2X3R in ipsilateral DRG) which might correlate with the observed pain alleviation. Our data indicate a role of HSP90 in MA pathophysiology, but further investigation is necessary to clarify the underlying mechanisms.

Spared nerve injury model to study orofacial pain.

BACKGROUND & OBJECTIVES: There are many difficulties in generating and testing orofacial pain in animal models. Thus, only a few and limited models that mimic the human condition are available. The aim of the present research was to develop a new model of trigeminal pain by using a spared nerve injury (SNI) surgical approach in the rat face (SNI-face). METHODS: Under anaesthesia, a small incision was made in the infraorbital region of adult male Wistar rats. Three of the main infraorbital nerve branches were tightly ligated and a 2 mm segment distal to the ligation was resected. Control rats were sham-operated by exposing the nerves. Chemical hyperalgesia was evaluated 15 days after the surgery by analyzing the time spent in face grooming activity and the number of head withdrawals in response to the orofacial formalin test. RESULTS: SNI-face rats presented a significant increase of the formalin-induced pain-related behaviours evaluated both in the acute and tonic phases (expected biphasic pattern), in comparison to sham controls. INTERPRETATION & CONCLUSIONS: The SNI-face model in the rat appears to be a valid approach to evaluate experimental trigeminal pain. Ongoing studies will test the usefulness of this model to evaluate therapeutic strategies for the treatment of orofacial pain.

Corticotropin-Releasing Factor Mediates Pain-Induced Anxiety through the ERK1/2 Signaling Cascade in Locus Coeruleus Neurons.

BACKGROUND: The corticotropin-releasing factor is a stress-related neuropeptide that modulates locus coeruleus activity. As locus coeruleus has been involved in pain and stress-related patologies, we tested whether the pain-induced anxiety is a result of the corticotropin-releasing factor released in the locus coeruleus. METHODS: Complete Freund's adjuvant-induced monoarthritis was used as inflammatory chronic pain model. α-Helical corticotropin-releasing factor receptor antagonist was microinjected into the contralateral locus coeruleus of 4-week-old monoarthritic animals. The nociceptive and anxiety-like behaviors, as well as phosphorylated extracellular signal-regulated kinases 1/2 and corticotropin-releasing factor receptors expression, were quantified in the paraventricular nucleus and locus coeruleus. RESULTS: Monoarthritic rats manifested anxiety and increased phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus and paraventricular nucleus, although the expression of corticotropin-releasing factor receptors was unaltered. α-Helical corticotropin-releasing factor antagonist administration reversed both the anxiogenic-like behavior and the phosphorylated extracellular signal-regulated kinases 1/2 levels in the locus coeruleus. CONCLUSIONS: Pain-induced anxiety is mediated by corticotropin-releasing factor neurotransmission in the locus coeruleus through extracellular signal-regulated kinases 1/2 signaling cascade.

Satellite glial cells in sensory ganglia: its role in pain / Células gliais satélite de gânglios sensitivos: o seu papel na dor / Células gliales satélite de ganglios sensoriales: su papel en el dolor

BACKGROUND AND OBJECTIVES: Satellite glial cells in sensory ganglia are a recent subject of research in the field of pain and a possible therapeutic target in the future. Therefore, the aim of this study was to summarize some of the important physiological and morphological characteristics of these cells and gather the most relevant scientific evidence about its possible role in the development of chronic pain. CONTENT: In the sensory ganglia, each neuronal body is surrounded by satellite glial cells forming distinct functional units. This close relationship enables bidirectional communication via a paracrine signaling between those two cell types. There is a growing body of evidence that glial satellite cells undergo structural and biochemical changes after nerve injury, which influence neuronal excitability and consequently the development and/or maintenance of pain in different animal models of chronic pain. CONCLUSIONS: Satellite glial cells are important in the establishment of physiological pain, in addition to being a potential target for the development of new pain treatments. .

JUSTIFICATIVA E OBJETIVOS: As células gliais satélite de gânglios sensitivos são um objeto recente de pesquisa na área da dor e um possível alvo terapêutico no futuro. Assim, este trabalho tem como objetivo resumir algumas das características morfológicas e fisiológicas mais importantes destas células e reunir as evidências científicas mais relevantes acerca do seu possível papel no desenvolvimento da dor crônica. CONTEÚDO: Nos gânglios sensitivos cada corpo neuronial é envolvido por células gliais satélite, formando unidades funcionais distintas. Esta íntima relação possibilita a comunicação bidirecional, através de uma sinalização parácrina, entre estes dois tipos de células. Existe um número crescente de evidências de que as células gliais satélite sofrem alterações estruturais e bioquímicas, após lesão nervosa, que influenciam a excitabilidade neuronial e consequentemente o desenvolvimento e/ou manutenção da dor, em diferentes modelos animais de dor crônica. CONCLUSÕES: As células gliais satélite são importantes no estabelecimento da dor não fisiológica e constituem um alvo potencial para o desenvolvimento de novos tratamentos da dor. .

JUSTIFICACIÓN Y OBJETIVOS: Las células gliales satélite de ganglios sensoriales son un objeto reciente de investigación en el área del dolor y un posible objeto terapéutico en el futuro. Por tanto, este trabajo intenta resumir algunas de las características morfológicas y fisiológicas más importantes de estas células y reunir las evidencias científicas más relevantes acerca de su posible papel en el desarrollo del dolor crónico. CONTENIDO: En los ganglios sensoriales cada cuerpo neuronal está envuelto por células gliales satélite, formando unidades funcionales distintas. Esta íntima relación posibilita la comunicación bidireccional a través de una señalización paracrina entre esos 2 tipos de células. Existe un número creciente de evidencias de que las células gliales satélite sufren alteraciones estructurales y bioquímicas después de la lesión nerviosa que influyen en la excitabilidad neuronal y por ende en el desarrollo y/o en el mantenimiento del dolor en diferentes modelos animales de dolor crónico. CONCLUSIONES: Las células gliales satélite son importantes en el establecimiento del dolor no fisiológico y son un potencial objetivo para el desarrollo de nuevos tratamientos del dolor. .

[Satellite glial cells in sensory ganglia: its role in pain]. / Células gliais satélite de gânglios sensitivos: o seu papel na dor.

BACKGROUND AND OBJECTIVES: Satellite glial cells in sensory ganglia are a recent subject of research in the field of pain and a possible therapeutic target in the future. Therefore, the aim of this study was to summarize some of the important physiological and morphological characteristics of these cells and gather the most relevant scientific evidence about its possible role in the development of chronic pain. CONTENT: In the sensory ganglia, each neuronal body is surrounded by satellite glial cells forming distinct functional units. This close relationship enables bidirectional communication via a paracrine signaling between those two cell types. There is a growing body of evidence that glial satellite cells undergo structural and biochemical changes after nerve injury, which influence neuronal excitability and consequently the development and/or maintenance of pain in different animal models of chronic pain. CONCLUSIONS: Satellite glial cells are important in the establishment of physiological pain, in addition to being a potential target for the development of new pain treatments.

Satellite glial cells surrounding primary afferent neurons are activated and proliferate during monoarthritis in rats: is there a role for ATF3?

Joint inflammatory diseases are debilitating and very painful conditions that still lack effective treatments. Recently, glial cells were shown to be crucial for the development and maintenance of chronic pain, constituting novel targets for therapeutic approaches. At the periphery, the satellite glial cells (SGCs) that surround the cell bodies of primary afferents neurons in the dorsal root ganglia (DRG) display hypertrophy, proliferation, and activation following injury and/or inflammation. It has been suggested that the expression of neuronal injury factors might initially trigger these SGCs-related events. We then aimed at evaluating if SGCs are involved in the establishment/maintenance of articular inflammatory pain, by using the monoarthritis (MA) model, and if the neuronal injury marker activating transcriptional factor 3 (ATF3) is associated with these SGCs' reactive changes. Western Blot (WB) analysis of the glial fibrillary acidic protein (GFAP) expression was performed in L4-L5 DRGs from control non-inflamed rats and MA animals at different time-points of disease (4, 7, and 14d, induced by complete Freund's adjuvant injection into the left hind paw ankle joint). Data indicate that SGCs activation is occurring in MA animals, particularly after day 7 of disease evolution. Additionally, double-immunostaining for ATF3 and GFAP in L5 DRG sections shows that SGCs's activation significantly increases around stressed neurons at 7d of disease, when compared with control animals. The specific labelling of GFAP in SGCs rather than in other cell types was also confirmed by immunohistochemical labeling. Finally, BrdU incorporation indicates that proliferation of SGCs is also significantly increased after 7 days of MA. Data indicate that SGCs play an important role in the mechanisms of articular inflammation, with 7 days of disease being a critical time-point in the MA model, and suggest that ATF3 might be involved in SGCs' reactive changes such as activation.

Neuronal injury marker ATF-3 is induced in primary afferent neurons of monoarthritic rats.

Activating transcription factor 3 (ATF-3) expression has been associated with several signaling pathways implicated in cellular stress response in many cell types and is usually regarded as a neuronal damage marker in dorsal root ganglia (DRG). We investigated ATF-3 expression in primary afferents in the monoarthritic (MA) model of chronic inflammatory joint pain. Immunohistochemistry revealed that ATF-3 is highly induced mainly in small and medium neurons, especially at 2 and 4 days of MA in L(5) DRGs. Colocalization with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4) demonstrated that ATF-3-immunoreactive cells are mainly peptidergic. The lack of significant differences in ATF-3 and pAkt colocalization indicated that ATF-3 is probably not involved in a pAkt-mediated survival pathway. Anti-inflammatory (ketoprofen) administration failed to reverse ATF-3 induction in MA rats, but significantly increased CGRP expression. These data suggest that ATF-3 expression is definitely involved in MA, actually marking injured neurons. Some degree of neuronal damage seems to occur right from the first days of disease, mainly affecting small-to-medium peptidergic neurons. The intra-articular injection of complete Freund's adjuvant and the generation of a neuroinflammatory environment seem to be the plausible explanation for the local nerve damage.

Nociceptive behaviour upon modulation of mu-opioid receptors in the ventrobasal complex of the thalamus of rats.

The role of mu-opioid receptors (MORs) in the inflammatory pain processing mechanisms within the ventrobasal complex of the thalamus (VB) is not well understood. This study investigated the effect of modulating MOR activity upon nociception, by stereotaxically injecting specific ligands in the VB. Nociceptive behaviour was evaluated in two established animal models of inflammatory pain, by using the formalin (acute and tonic pain) and the ankle-bend (chronic monoarthritic pain) tests. Control (saline intra-VB injection) formalin-injected rats showed acute and tonic pain-related behaviours. In contrast, intrathalamic administration of [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin acetate (DAMGO), a MOR-specific agonist, induced a statistically significant decrease of all tonic phase pain-related behaviours assessed until 30-35min after formalin hind paw injection. In the acute phase only the number of paw-jerks was affected. In monoarthritic rats, there was a noticeable antinociceptive effect with approximately 40min of duration, as denoted by the reduced ankle-bend scores observed after DAMGO injection. Intra-VB injection of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP), a specific MOR antagonist, or of CTOP followed, 10min after, by DAMGO had no effects in either formalin or ankle-bend tests. Data show that DAMGO-induced MOR activation in the VB has an antinociceptive effect in the formalin test as well as in chronic pain observed in MA rats, suggesting an important and specific role for MORs in the VB processing of inflammatory pain.

Delta opioid receptor mRNA expression is changed in the thalamus and brainstem of monoarthritic rats.

Changes in the mRNA expression of neurotransmitters receptors under chronic pain conditions have been described in various areas of the central nervous system (CNS). Delta opioid receptors (DORs) have been implicated in pain mechanisms but, although its mRNA expression has been studied in the rat CNS, there are no reports describing its distribution in specific thalamic and brainstem nuclei during chronic inflammatory pain. Here, in situ hybridization for DOR mRNA was performed in brain sections from control and monoarthritic (MA) rats with 2, 4, 7 and 14 days of inflammation. Grain densities were determined bilaterally in the ventrobasal complex (VB), posterior (Po), centromedial/centrolateral (CM/CL) and reticular (Rt) nuclei of the thalamus, and in the dorsal reticular (DRt), lateral reticular (LRt) and parvocellular reticular (PCRt) nuclei of the brainstem. Control animals exhibited weak mRNA expression in the VB, Po and CM/CL, as well as in PCRt, while moderate grain densities were observed in the Rt, DRt and LRt. During MA, DOR mRNA expression was significantly decreased (22%) in the Rt contralateral to the affected joint at both 7 and 14 days of inflammation, as compared to controls. A bilateral reduction (35%) was also observed in the DRt at 14 days of MA, while a contralateral increase was found in the PCRt at 7 days (+39%). No significant changes were observed in the other regions analyzed. Thus, data show changes in the DOR mRNA expression during the development of chronic inflammatory pain, in thalamic and brainstem nuclei implicated in pain processing mechanisms.

Inhibition of pain behavior by GABA(B) receptors in the thalamic ventrobasal complex: effect on normal rats subjected to the formalin test of nociception.

The ventrobasal complex of the thalamus (VB) participates in the transmission and modulation of noxious information. Recent data suggested that GABA(B) receptors in the VB might be involved in the modulation of neuronal activity in response to chronic noxious input. However, in acute inflammatory pain, the role of GABA(B) receptors in the VB remains unknown. The formalin test of nociception was performed in rats stereotaxically injected in the VB contralateral to the formalin-injected paw, with saline (controls), baclofen (0.5 and 0.875 microg), a specific GABA(B) receptor agonist or CGP35348 (25 microg), a GABA(B) receptor antagonist. Control animals exhibited phase 1 (acute pain) and phase 2 (tonic pain) nociception-related activities as previously described. The higher dose of baclofen induced a significant decrease of all pain-related behaviors in both phases of the test and had no observable effects on the animals' motor function, while the lower dose could not reduce the total pain-related activities. Injection of CGP35348 prior to baclofen reduced the antinociceptive effect caused by baclofen during phase 2 in the paw-jerks and in total pain-related activities. CGP35348 alone had antinociceptive effects in both phases, though less pronounced than baclofen 0.875 microg in the total pain-related activities during phase 2. Data demonstrate that both the blockade and the activation of GABA(B) receptors in the VB of rats induce antinociception in acute and tonic pain. An important role for GABA(B) receptors on the thalamic processing of nociceptive input in the VB is suggested.

Administration of baclofen, a gamma-aminobutyric acid type B agonist in the thalamic ventrobasal complex, attenuates allodynia in monoarthritic rats subjected to the ankle-bend test.

gamma-Aminobutyric acid type B (GABAB) receptors are involved in the modulation of neuronal activity in response to chronic noxious input. However, the effect of their activation in chronic inflammatory pain in relay thalamic nuclei such as the ventrobasal complex (VB) is not known. In this study, experimental groups of 2, 4, and 14 days monoarthritic (MA) rats were injected with saline (controls) or baclofen (0.875 microg), a specific GABAB receptor agonist, in the VB contralateral to the inflamed joint, and the ankle-bend test was performed. Ankle-bend scores in control animals were near the maximum and were rather constant throughout the entire experimental period, indicating severe nociception. The same was observed in 2 days MA rats injected with baclofen. In the 4 days MA group, the response to baclofen injection was inconsistent among different animals, whereas, in 14 days MA rats, baclofen caused clear antinociceptive effects. Additionally, a 0.5 microg dose of baclofen was tested in 14 days MA rats, but no effect was observed, whereas a 1.25 mug dose produced visible side effects. Baclofen injections that did not target the VB but reached neighboring nuclei were ineffective in reducing nociception. Data demonstrate that the activation of the GABAB receptors by baclofen in the VB of MA rats leads to a decrease of nociception. Moreover, the response depends on the time course of the disease, suggesting the occurrence of different excitatory states of thalamic VB neurons. In conclusion, GABAB receptors in the VB play an important role in chronic inflammatory pain processing.