µ-Opioid Receptor Activation at the Dorsal Reticular Nucleus Shifts Diffuse Noxious Inhibitory Controls to Hyperalgesia in Chronic Joint Pain in Male Rats.

BACKGROUND: The dorsal reticular nucleus is a pain facilitatory area involved in diffuse noxious inhibitory control (DNIC) through opioidergic mechanisms that are poorly understood. The hypothesis was that signaling of µ-opioid receptors is altered in this area with prolonged chronic inflammatory pain and that this accounts for the loss of DNICs occurring in this condition. METHODS: Monoarthritis was induced in male Wistar rats (n = 5 to 9/group) by tibiotarsal injection of complete Freund's adjuvant. The immunolabeling of µ-opioid receptors and the phosphorylated forms of µ-opioid receptors and cAMP response element binding protein was quantified. Pharmacologic manipulation of µ-opioid receptors at the dorsal reticular nucleus was assessed in DNIC using the Randall-Selitto test. RESULTS: At 42 days of monoarthritis, µ-opioid receptor labeling decreased at the dorsal reticular nucleus, while its phosphorylated form and the phosphorylated cAMP response element binding protein increased. [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate (DAMGO) enhanced DNIC analgesia in normal animals (means ± SD: pre-DNIC: 126.9 ± 7.0 g; DNIC - DAMGO: 147.5 ± 8.0 g vs. DNIC + DAMGO: 198.1 ± 19.3 g; P < 0.001), whereas it produced hyperalgesia in monoarthritis (pre-DNIC: 67.8 ± 7.5 g; DNIC - DAMGO: 70.6 ± 7.7 g vs. DNIC + DAMGO: 32.2 ± 2.6 g; P < 0.001). An ultra-low dose of naloxone, which prevents the excitatory signaling of the µ-opioid receptor, restored DNIC analgesia in monoarthritis (DNIC - naloxone: 60.0 ± 6.1 g vs. DNIC + naloxone: 98.0 ± 13.5 g; P < 0.001), compared to saline (DNIC - saline: 62.5 ± 5.2 g vs. DNIC + saline: 64.2 ± 3.8 g). When injected before DAMGO, it restored DNIC analgesia and decreased the phosphorylated cAMP response element binding protein in monoarthritis. CONCLUSIONS: The dorsal reticular nucleus is likely involved in a facilitatory pathway responsible for DNIC hyperalgesia. The shift of µ-opioid receptor signaling to excitatory in this pathway likely accounts for the loss of DNIC analgesia in monoarthritis.

Diffuse noxious inhibitory controls in chronic joint inflammatory Pain: Study of the descending serotonergic modulation mediated through 5HT3 receptors.

The loss of diffuse noxious inhibitory controls (DNIC) is recognized as a predictor of chronic pain. Mechanistically, DNIC produces analgesia by a heterotopically applied conditioning-noxious stimulus (CS) and yet underexplored descending modulatory inputs. Here, we aimed at studying DNIC in monoarthritis (MA) by exploring the spinal component of the descending serotonergic system, specifically 5-hydroxytryptamine 3 receptors (5-HT3R). MA was induced in male Wistar rats by tibiotarsal injection of complete Freund's adjuvant. Mechanical hyperalgesia and DNIC were assessed weekly by the Randall-Selitto test. Immunohistochemistry was used to quantify spinal 5-HT3R, and tryptophan hydroxylase (TPH) colocalization with phosphorylated extracellular signal-regulated protein kinases 1/2 at the rostroventromedial medulla (RVM). Spinal serotonin (5-HT) was quantified by HPLC. The effects of intrathecal ondansetron, a 5-HT3R antagonist, were assessed on mechanical hyperalgesia and DNIC. MA resulted in a prolonged steady-state mechanical hyperalgesia. In contrast, DNIC peaked after 28 days, decreasing afterwards until extinction at 42 days. At this later timepoint, MA rats showed increased: (i) spinal 5-HT3R and 5-HT levels, (ii) neuronal serotonergic activation and TPH expression at the RVM. Ondansetron reversed mechanical hyperalgesia and restored DNIC, regardless of being administered before or after CS. However, data variability was higher upon administration before CS in MA-animals. Prolonged MA upregulates the descending serotonergic modulation, which simultaneously results in increased nociception and DNIC extinction, through 5-HT3R. Our data suggest a role for spinal 5-HT3R in the top-down modulation of DNIC. Additionally, these receptors may also be involved in the bottom-up circuitry implicated in the trigger of DNIC.

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.

Attenuation of the Diffuse Noxious Inhibitory Controls in Chronic Joint Inflammatory Pain Is Accompanied by Anxiodepressive-Like Behaviors and Impairment of the Descending Noradrenergic Modulation.

The noradrenergic system is paramount for controlling pain and emotions. We aimed at understanding the descending noradrenergic modulatory mechanisms in joint inflammatory pain and its correlation with the diffuse noxious inhibitory controls (DNICs) and with the onset of anxiodepressive behaviours. In the complete Freund's adjuvant rat model of Monoarthritis, nociceptive behaviors, DNICs, and anxiodepressive-like behaviors were evaluated. Spinal alpha2-adrenergic receptors (a2-AR), dopamine beta-hydroxylase (DBH), and noradrenaline were quantified concomitantly with a2-AR pharmacologic studies. The phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) were quantified in the Locus coeruleus (LC), amygdala, and anterior cingulate cortex (ACC). DNIC was attenuated at 42 days of monoarthritis while present on days 7 and 28. On day 42, in contrast to day 28, noradrenaline was reduced and DBH labelling was increased. Moreover, spinal a2-AR were potentiated and no changes in a2-AR levels were observed. Additionally, at 42 days, the activation of ERKs1/2 was increased in the LC, ACC, and basolateral amygdala. This was accompanied by anxiety- and depressive-like behaviors, while at 28 days, only anxiety-like behaviors were observed. The data suggest DNIC is attenuated in prolonged chronic joint inflammatory pain, and this is accompanied by impairment of the descending noradrenergic modulation and anxiodepressive-like behaviors.

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.

Glial activation in the collagenase model of nociception associated with osteoarthritis.

Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3-L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord. Inhibition of glial cell activation by fluorocitrate decreases these osteoarthritis-associated nociceptive behaviours. These results suggest that glial cell activation may play a role in the development of chronic pain in this experimental model of osteoarthritis.

Activation of Extracellular Signal-Regulated Kinases (ERK 1/2) in the Locus Coeruleus Contributes to Pain-Related Anxiety in Arthritic Male Rats.

Background: There is increasing evidence suggesting that the Locus Coeruleus plays a role in pain-related anxiety. Indeed, we previously found that prolonged arthritis produces anxiety-like behavior in rats, along with enhanced expression of phosphorylated extracellular signal-regulated kinase 1/2 (a marker of plasticity) in the Locus Coeruleus. However, it is unknown how this effect correlates with the electrophysiological activity of Locus Coeruleus neurons or pain-related anxiety. Methods: Using the complete Freund's adjuvant model of monoarthritis in male Sprague-Dawley rats, we studied the behavioral attributes of pain and anxiety as well as Locus Coeruleus electrophysiology in vivo 1 (MA1W) and 4 weeks (MA4W) after disease induction. Results: The manifestation of anxiety in MA4W was accompanied by dampened tonic Locus Coeruleus activity, which was coupled to an exacerbated evoked Locus Coeruleus response to noxious stimulation of the inflamed and healthy paw. When a mitogen-activating extracellular kinase inhibitor was administered to the contralateral Locus Coeruleus of MA4W, the phosphorylated extracellular signal-regulated kinase 1/2 levels in the Locus Coeruleus were restored and the exaggerated evoked response was blocked, reversing the anxiogenic-like behavior while pain hypersensitivity remained unaltered. Conclusion: As phosphorylated extracellular signal-regulated kinase 1/2 blockade in the Locus Coeruleus relieved anxiety and counteracted altered LC function, we propose that phosphorylated extracellular signal-regulated kinase 1/2 activation in the Locus Coeruleus plays a crucial role in pain-related anxiety.

Noradrenergic Locus Coeruleus pathways in pain modulation.

The noradrenergic system is crucial for several activities in the body, including the modulation of pain. As the major producer of noradrenaline (NA) in the central nervous system (CNS), the Locus Coeruleus (LC) is a nucleus that has been studied in several pain conditions, mostly due to its strategic location. Indeed, apart from a well-known descending LC-spinal pathway that is important for pain control, an ascending pathway passing through this nucleus may be responsible for the noradrenergic inputs to higher centers of the pain processing, such as the limbic system and frontal cortices. Thus, the noradrenergic system appears to modulate different components of the pain experience and accordingly, its manipulation has distinct behavioral outcomes. The main goal of this review is to bring together the data available regarding the noradrenergic system in relation to pain, particularly focusing on the ascending and descending LC projections in different conditions. How such findings influence our understanding of these conditions is also discussed.

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.

ERK1/2: Function, signaling and implication in pain and pain-related anxio-depressive disorders.

Despite the increasing knowledge regarding pain modulation, the understanding of the mechanisms behind a complex and pathologic chronic pain condition is still insufficient. These knowledge gaps might result in ineffective therapeutic approaches to relieve painful sensations. As a result, severe untreated chronic pain frequently triggers the onset of new disorders such as depression and/or anxiety, and therefore, both the diagnosis and treatment of patients suffering from chronic pain become seriously compromised, prompting a self-perpetuating cycle of symptomatology. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are molecules strongly implicated in the somatic component of pain at the spinal cord level and have been emerging as mediators of the emotional-affective component as well. Although these molecules might represent good biomarkers, their use as pharmacological targets is still open to discussion as paradoxical information has been obtained. Here we review the current scientific literature regarding ERK1/2 signaling in the modulation of pain, depression and anxiety, including the emotional-affective spheres of the pain experience.

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.

Reversal of monoarthritis-induced affective disorders by diclofenac in rats.

BACKGROUND: Nonsteroidal anti-inflammatory drugs are effective for arthritic pain, but it is unknown whether they also benefit anxiety and depression that frequently coexist with pain. Using the monoarthritis model, the authors evaluated the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in structures implicated in both sensorial and emotional pain spheres, and it was verified whether analgesia can reverse monoarthritis-mediated affective responses. METHODS: Monoarthritis was induced in male rats by complete Freund's adjuvant injection. Allodynia (ankle-bend test), mechanical hyperalgesia (paw-pinch test), anxiety- and depression-like behaviors (elevated zero maze and forced swimming tests, respectively), and ERK1/2 phosphorylation (Western blot) in the spinal cord, paragigantocellularis nucleus, locus coeruleus, and prefrontal cortex were evaluated at 4, 14, and 28 days postinoculation (n = 6 per group). Changes in these parameters were evaluated after induction of analgesia by topical diclofenac (n = 5 to 6 per group). RESULTS: Despite the pain hypersensitivity and inflammation throughout the testing period, chronic monoarthritis (28 days) also resulted in depressive- (control [mean ± SEM]: 38.3 ± 3.7 vs. monoarthritis: 51.3 ± 2.0; P < 0.05) and anxiogenic-like behaviors (control: 36.8 ± 3.7 vs. monoarthritis: 13.2 ± 2.9; P < 0.001). These changes coincided with increased ERK1/2 activation in the spinal cord, paragigantocellularis, locus coeruleus, and prefrontal cortex (control vs. monoarthritis: 1.0 ± 0.0 vs. 5.1 ± 20.8, P < 0.001; 0.9 ± 0.0 vs. 1.9 ± 0.4, P < 0.05; 1.0 ± 0.3 vs. 2.9 ± 0.6, P < 0.01; and 1.0 ± 0.0 vs. 1.8 ± 0.1, P < 0.05, respectively). Diclofenac decreased the pain threshold of the inflamed paw and reversed the anxio-depressive state, restoring ERK1/2 activation levels in the regions analyzed. CONCLUSION: Chronic monoarthritis induces affective disorders associated with ERK1/2 phosphorylation in paragigantocellularis, locus coeruleus, and prefrontal cortex which are reversed by diclofenac analgesia. (Anesthesiology 2014; 120:1476-90).

Intra-articular injection of collagenase in the knee of rats as an alternative model to study nociception associated with osteoarthritis.

INTRODUCTION: Animal models currently used in osteoarthritis-associated pain research inadequately reproduce the initiating events and structural pathology of human osteoarthritis. Conversely, intra-articular injection of collagenase is a structurally relevant model, as it induces articular degeneration both by digesting collagen from cartilage and by causing articular instability, thereby reproducing some of the main events associated with osteoarthritis onset and development. Here, we evaluated if the intra-articular injection of collagenase can be an alternative model to study nociception associated with osteoarthritis. METHODS: Osteoarthritis was induced by two intra-articular injections of either 250 U or 500 U of collagenase into the left knee joint of adult male Wistar rats. A six weeks time-course assessment of movement- and loading-induced nociception was performed by the Knee-Bend and CatWalk tests. The effect of morphine, lidocaine and diclofenac on nociceptive behaviour was evaluated in animals injected with 500 U of collagenase. Joint histopathology was scored for both doses throughout time. The expression of transient receptor potential vanilloid 1 (TRPV1) in ipsilateral dorsal root ganglia (DRG) was evaluated. RESULTS: An increase in nociceptive behaviour associated with movement and loading of affected joints was observed after intra-articular collagenase injection. With the 500 U dose of collagenase, there was a significant correlation between the behavioural and the histopathological osteoarthritis-like structural changes developed after six weeks. One week after injection of 500 U collagenase, swelling of the injected knee and inflammation of the synovial membrane were also observed, indicating the occurrence of an early inflammatory reaction. Behavioural changes induced by the 500 U dose of collagenase were overall effectively reversed by morphine and lidocaine. Diclofenac was effective one week after injection. TRPV1 expression increased six weeks after 500 U collagenase injection. CONCLUSION: We conclude that the intra-articular injection of 500 U collagenase in the knee of rats can be an alternative model for the study of nociception associated with osteoarthritis, since it induces significant nociceptive alterations associated with relevant osteoarthritis-like joint structural changes.

Social stress exacerbates the aversion to painful experiences in rats exposed to chronic pain: the role of the locus coeruleus.

Stressful experiences seem to negatively influence pain perception through as yet unknown mechanisms. As the noradrenergic locus coeruleus (LC) nucleus coordinates many components of the stress response, as well as nociceptive transmission, we evaluated whether the sensory and affective dimension of chronic neuropathic pain worsens in situations of stress due to adaptive changes of LC neurons. Accordingly, male rats were socially isolated for 5 weeks, and in the last 2 weeks, neuropathic pain was induced by chronic constriction injury. In this situation of stress, chronic pain selectively heightened the animal's aversion to painful experiences (affective pain), as measured in the place escape/avoidance test, although no changes were observed in the sensory dimension of pain. In addition, electrophysiological recordings of LC neurons showed a low tonic but exacerbated nociceptive-evoked activity when the injured paw was stimulated. These changes were accompanied by an increase in tyrosine hydroxylase and gephyrin expression in the LC. Furthermore, intra-LC administration of bicuculline, a γ-aminobutyric acid-A receptor antagonist, attenuated the negative affective effects of pain. These data show that changes in the LC are greater than those expected from the simple summation of each independent factor (pain and stress), revealing mechanisms through which stressors may exacerbate pain perception without affecting the sensorial dimension.

The function of alpha-2-adrenoceptors in the rat locus coeruleus is preserved in the chronic constriction injury model of neuropathic pain.

RATIONALE: Peripheral neuropathic pain is a chronic condition that may produce plastic changes in several brain regions. The noradrenergic locus coeruleus (LC) is a crucial component of ascending and descending pain pathways, both of which are frequently compromised after nerve injury. OBJECTIVES: The objective of the study was to examine whether chronic constriction injury (CCI), a model of neuropathic pain, alters noradrenergic activity in the rat LC. METHODS: Activity in the LC was assessed by electrophysiology and microdialysis, while protein expression was monitored in western blots and by immunohistochemistry. RESULTS: The pain threshold had dropped in injured rats 7 days after inducing neuropathy. While alpha-2-adrenoceptors mediate activity in the LC and in its terminal areas, no alterations in either spontaneous neuronal activity or extracellular noradrenaline levels were observed following CCI. Moreover, alpha-2-adrenoceptor activity in the LC of CCI rats remained unchanged after systemic administration of UK14,304, RX821002 or desipramine. Accordingly, extracellular noradrenaline levels in the LC were similar in CCI and control animals following local administration of clonidine or RX821002. In addition, there were no changes in the expression of the alpha-2-adrenoceptors, Gαi/z subunits or the regulators of G-protein signaling. However, pERK1/2 (phosphorylated extracellular signal-regulated kinases 1/2) expression augmented in the spinal cord, paragigantocellularis nucleus (PGi) and dorsal raphe nucleus (DRN) following CCI. CONCLUSIONS: Neuropathic pain is not accompanied by modifications in tonic LC activity after the onset of pain. This may indicate that the signals from the PGi and DRN, the excitatory and inhibitory afferents of the LC, cancel one another out.