Activation Of Spinal Glia Research Articles

Spinal pain processing in arthritis: Neuron and glia (inter)actions.

Diseases of joints are among the most frequent causes of chronic pain. In the course of joint diseases, the peripheral and the central nociceptive system develop persistent hyperexcitability (peripheral and central sensitization). This review addresses the mechanisms of spinal sensitization evoked by arthritis. Electrophysiological recordings in anesthetized rats from spinal cord neurons with knee input in a model of acute arthritis showed that acute spinal sensitization is dependent on spinal glutamate receptors (AMPA, NMDA, and metabotropic glutamate receptors) and supported by spinal actions of neuropeptides such as neurokinins and CGRP, by prostaglandins, and by proinflammatory cytokines. In several chronic arthritis models (including immune-mediated arthritis and osteoarthritis) spinal glia activation was observed to be coincident with behavioral mechanical hyperalgesia which was attenuated or prevented by intrathecal application of minocycline, fluorocitrate, and pentoxyfylline. Some studies identified specific pathways of micro- and astroglia activation such as the purinoceptor- (P2 X7 -) cathepsin S/CX3 CR1 pathway, the mobility group box-1 protein (HMGB1), and toll-like receptor 4 (TLR4) activation, spinal NFκB/p65 activation and others. The spinal cytokines TNF, interleukin-6, interleukin-1β, and others form a functional spinal network characterized by an interaction between neurons and glia cells which is required for spinal sensitization. Neutralization of spinal cytokines by intrathecal interventions attenuates mechanical hyperalgesia. This effect may in part result from local suppression of spinal sensitization and in part from efferent effects which attenuate the inflammatory process in the joint. In summary, arthritis evokes significant spinal hyperexcitability which is likely to contribute to the phenotype of arthritis pain in patients.

Participation of transient receptor potential vanilloid 1 in the analgesic effect of duloxetine for paclitaxel induced peripheral neuropathic pain

Painful peripheral neuropathy is a common dose-limiting side effect of chemotherapeutic paclitaxel (PTX) treatment. The American Society of Clinical Oncology (ASCO) recommends duloxetine (DUL) as a promising treatment alternative for chemotherapy-induced peripheral neuropathic pain. However, this recommendation lacks a robust theoretical basis and supporting data. To elucidate the involvement of transient receptor potential vanilloid 1 (TRPV1) in the analgesic effect of DUL for PTX-induced neuropathic pain, TRPV1 expression in the lumbar dorsal root ganglion (DRG) and spinal cord was evaluated following intraperitoneal administration of PTX (2 mg/kg/day) for four alternate days in rats. Western blot and immunohistochemistry suggested that a cumulative dosage of PTX (8 mg/kg) upregulated TRPV1 expression in the lumbar DRG and spinal dorsal horn (SDH) at day 14 post treatment. TRPV1 upregulation in the DRG was mainly expressed in calcitonin gene-related peptide (CGRP) and IB-4 positive small-size sensory neurons. Additionally, PTX increased CGRP and substance P (SP) expression in the DRG and SDH, induced SDH microglia and astrocyte activation, and upregulated spinal tumor necrosis factor-α (TNF-α) but not IL-1β or IL-10 expression. Behavioral detection showed that PTX-related mechanical and thermal hyperalgesia was significantly inhibited by consecutive administration of DUL 20 mg/kg/day greater than 10 mg/kg/day for 5 days starting at day 10 post PTX injection. Furthermore, DUL (20 mg/kg/day) for 5 days markedly ameliorated PTX-induced TRPV1, CGRP, and SP upregulation in the DRG and SDH, and mitigated PTX-induced spinal cord glia activation and TNF-α expression. Moreover, the pharmacological blockade of TRPV1 resulted in an analgesic effect on PTX-induced hyperalgesia. Collectively, these results suggest that DUL alleviates PTX-induced peripheral neuropathic pain by suppressing TRPV1 upregulation in the lumbar DRG and SDH, which is followed by a reduction in CGRP and SP release, as well as spinal glia activation and TNF-α expression.

Electroacupuncture Alleviates Paclitaxel-Induced Peripheral Neuropathic Pain in Rats via Suppressing TLR4 Signaling and TRPV1 Upregulation in Sensory Neurons.

Paclitaxel-induced peripheral neuropathy is a common adverse effect during paclitaxel treatment resulting in sensory abnormalities and neuropathic pain during chemotherapy and in cancer survivors. Conventional therapies are usually ineffective and possess adverse effects. Here, we examined the effects of electroacupuncture (EA) on a rat model of paclitaxel-induced neuropathic pain and related mechanisms. EA robustly and persistently alleviated paclitaxel-induced pain hypersensitivities. Mechanistically, TLR4 (Toll-Like Receptor 4) and downstream signaling MyD88 (Myeloid Differentiation Primary Response 88) and TRPV1 (Transient Receptor Potential Vallinoid 1) were upregulated in dorsal root ganglion (DRGs) of paclitaxel-treated rats, whereas EA reduced their overexpression. Ca2+ imaging further indicated that TRPV1 channel activity was enhanced in DRG neurons of paclitaxel-treated rats whereas EA suppressed the enhanced TRPV1 channel activity. Pharmacological blocking of TRPV1 mimics the analgesic effects of EA on the pain hypersensitivities, whereas capsaicin reversed EA’s effect. Spinal astrocytes and microglia were activated in paclitaxel-treated rats, whereas EA reduced the activation. These results demonstrated that EA alleviates paclitaxel-induced peripheral neuropathic pain via mechanisms possibly involving suppressing TLR4 signaling and TRPV1 upregulation in DRG neurons, which further result in reduced spinal glia activation. Our work supports EA as a potential alternative therapy for paclitaxel-induced neuropathic pain.

The Effects of Generally Administered Anti–Nerve Growth Factor Receptor (p75NTR) Antibody on Pain-Related Behavior, Dorsal Root Ganglia, and Spinal Glia Activation in a Rat Model of Brachial Plexus Avulsion

To investigate the effect of intraperitoneal administration of an anti-p75 neurotrophin receptor (p75NTR) antibody on reducing neuropathic pain in a rat model of brachial plexus avulsion (BPA). We randomly assigned 40 male Wistar rats to 4 groups. In the BPA group, the C8-T1 roots were avulsed from the spinal cord at the lower trunk level, and saline was administered intraperitoneally. In the anti-p75NTR groups, 1 μL or 50 μL anti-p75NTR antibody was administered intraperitoneally after avulsion. In the sham-operated group, the lower trunk level was exposed, and saline was administered intraperitoneally. Mechanical hyperalgesia and pain-induced walking patterns were measured using von Frey filaments and CatWalk gait analysis at various time points until 15 days after administration. At 3 and 15 days after administration, sensory neurons involved in pain perception and satellite glial cells in the ipsilateral C7 dorsal root ganglia were immunolabeled with antibodies against calcitonin gene-related peptide and glial fibrillary acidic protein (GFAP), respectively. At both time points, microglial and astrocyte activation, indicative of spinal pain transmission, were immunohistochemically examined in the ipsilateral dorsal horn of the spinal cord (C7) using anti-ionized calcium-binding adaptor molecule 1 and anti-GFAP antibodies, respectively. The gait pattern was significantly improved in both anti-p75NTR groups compared with the BPA group. There were significantly fewer calcitonin gene-related peptide-immunoreactive (IR) neurons, neurons encircled by GFAP-IR satellite glial cells, and GFAP-IR astrocytes in both anti-p75NTR groups compared with the BPA group at both time points. Fewer ionized calcium-binding adaptor molecule 1-IR microglia were quantified in both anti-p75NTR groups compared with the BPA group, but this was only significant at 15 days after administration. Systemic application of the p75NTR inhibitory antibody suppressed neuropathic pain after BPA. p75NTR may be a potential therapeutic target for the clinical treatment of neuropathic pain in BPA injury.

Heme Induced Spinal Microglial Cell Activation By TLR4 and Endoplasmic Reticulum Stress in Sickle Mice

Sickle cell disease (SCD) is associated with pain, which remains a major challenge to treat. Earlier, we showed that peripheral mechanisms including mast cell activation in the skin contribute to pain in sickle mice (Vincent et al., Blood 2013). Mast cell activation in sickle mice was accompanied by a significant increase in toll-like receptor 4 (TLR4) as compared to mast cells from control mice. Since peripheral as well as central mechanisms are involved in nociception, we examined the central mechanisms underlying pain in SCD. TLR4 signaling is involved in inflammatory and neuropathic pain (Wang et al., FASEB 2013 and Hutchinson et al., Eur J Neurosci 2008). Microglial cells, the “macrophages” of the central nervous system in the spinal cord are critically involved in the development and maintenance of pain. Binding of an endogenous ligand to TLR4 is an important step in the regulation of microglial activity in pain facilitation. We hypothesized that heme, released during hemolysis in SCD, is a ligand for TLR4 expressed on spinal microglia.Methods. We isolated microglial cells from the spinal cords of HbSS-BERK (sickle) and HbAA-BERK (control) mice. To assess mitochondrial activity, we analyzed reactive oxygen species (ROS) and ATP, since increased ROS and decreased ATP are suggestive of mitochondrial dysfunction, which in turn is influenced by endoplasmic reticulum (ER) stress. ROS in the microglial cells was determined by utilizing the cell permeable reagent 2’,7’-dichlorofluorescein, which is oxidized by ROS to form a fluorescent compound, with the max excitation and emission spectra of 495 nm and 529 nm, respectively. ATP production was measured by a luminescence based assay from PerkinElmer (ATPlite).Results. Stimulation of microglia from control and sickle mice with hemin in vitro led to a several-fold increase in TLR4 gene transcripts in a time-dependent manner. Additionally, hemin induced the production of proinflammatory cytokines, TNF-α and IL-6, and ROS compared to vehicle-treated microglial cells from both sickle and control mice (p<0.01 for both). TAK-242 and LPS-RS, inhibitors of TLR4, ameliorated hemin-induced ROS production in microglial cells (p<0.01 and p<0.001 vs. hemin, respectively). Microglial cells treated with hemin showed a significant reduction in ATP content (p<0.01 vs. vehicle). Furthermore, hemin treatment increased expression of the ER stress protein, XBP1, in sickle and control microglial cells (40% increase in the expression of XBP1 compared to unstimulated), which was attenuated by the TLR4 inhibitor, LPS-RS (30% decrease compared to hemin stimulated), suggesting that hemin-induced TLR4 activation leads to ER stress. The ER stress inhibitor, salubrinal, attenuated hemin-induced ROS production from microglial cells (p<0.01 vs. vehicle). Moreover, hemin significantly stimulated the phosphorylation of p38MAPK, Stat3, Akt and MAPK/ERK in a time-dependent manner in both control and sickle glial cells. Whole spinal cord lysates from sickle mice showed significantly higher density of protein bands for phosphorylated p38MAPK, Stat3, Akt and MAPK/ERK, as compared to those from control mice, indicative of ongoing heme-induced glial activation and nociceptive signaling in spinal cords of sickle mice. Complementary to nociceptive signaling, ROS was significantly higher in sickle as compared to control mice spinal cords (p<0.05). Since hemin activates glial cells from control mice, it is a likely cause of microglial activation in sickle mice and because it further augments activation of glial cells from sickle mice, it may lead to a sustained activation of spinal glia. Therefore, hemin induces ER stress via activation of TLR4 resulting in the generation of ROS, oxidative stress and inflammation leading to the activation of microglial cells, which in turn release mediators that excite and sensitize spinal nociceptive neurons, thus maintaining chronic pain. These data suggest that inhibitors of TLR4 and ER stress may be of therapeutic benefit in treating pain in SCD. DisclosuresNo relevant conflicts of interest to declare.

Effects of intraperitoneal thymoquinone on chronic neuropathic pain in rats.

In this study, we evaluated the protective effects of thymoquinone, the major constituent of Nigella sativa seeds on the neuropathic pain of rats with chronic constrictive injury of the sciatic nerve. Rats received repeated administration of thymoquinone (1.25, 2.5, and 5 mg/kg, i. p.) once a day for 14 days, beginning immediately after the nerve injury. Mechanical allodynia, cold allodynia, and thermal hyperalgesia were assessed with the von Frey filament, acetone drop, or radiant heat stimulus, respectively. Recent evidence points towards a role of oxidative stress, spinal glia activation, and cell death in the pathogenesis of neuropathic pain. Ionized calcium-binding adapter molecule 1 (a marker of microglia), glial fibrillary acidic protein (a marker of astroglia), Bcl2-associated X protein (a proapoptotic protein), and B-cell lymphoma protein 2 (an antiapoptotic protein) were measured using Western blot on days 3, 7, and 14 post chronic constrictive injury. The changes in the protein levels of malondialdehyde and glutathione, biomarkers of oxidative stress, were assessed by spectrophotometric assay on day 14 post chronic constrictive injury. Repeated treatment with thymoquinone (2.5 and 5 mg/kg) significantly alleviated behavioral signs of neuropathic pain. In the lumbar spinal cord of neuropathic rats, ionized calcium-binding adapter molecule 1 and Bcl2-associated X protein increased on day 3 post chronic constrictive injury, whereas B-cell lymphoma protein 2 did not significantly change. After repeated thymoquinone administration, the elevated Bcl2-associated X protein and ionized calcium-binding adapter molecule reduced on day 3, while the level of B-cell lymphoma protein 2 was even stimulated. Ionized calcium-binding adapter molecule and Bcl2-associated X protein/B-cell lymphoma protein 2 ratio declined by days 7 and 14; consequently, there were no significant differences among groups. No or little change was observed in the glial fibrillary acidic protein content during the study. Chronic constrictive injury produced a significant increase in the levels of malondialdehyde and decrease in the contents of glutathione on day 14. Thymoquinone treatment (2.5 and 5 mg/kg) restored the levels of malondialdehyde. High dose of thymoquinone (5 mg/kg) also reversed the decreased glutathione in the injured animals. Our results indicate that, microglia, apoptotic factors, and oxidative stress rather than astroglia contribute to the pathogenesis of chronic constrictive injury, and thymoquinone plays an anti-nociceptive role possibly by antioxidant effects and inhibition of microglia activity.

Effects of Anti-NGF Receptor (p75NTR) Antibody on Nociceptive Behavior and Spinal Glia Activation in the Rat Brachial Plexus Avulsion Model (Second Report): N/A - Not a clinical study

We hypothesized that inhibition of the p75 neurotrophin receptor (p75NTR), the low-affinity NGF receptor and common neurotrophin receptor, reduces neuropathic pain in the rat brachial plexus avulsion (BPA) model.

Resveratrol attenuates bone cancer pain through the inhibition of spinal glial activation and CX3CR1 upregulation.

The present study examined the effects of intrathecal use of resveratrol on pain hypersensitivities, spinal glia activation, and CX3CR1 expression in the model of bone cancer pain (BCP). The BCP model was established through intrathecally injecting Walker 256 mammary gland carcinoma cells to Sprague-Dawley rats. We found that spinal CX3CR1 expression and glial activation aggravated after inoculation. Resveratrol (i.t.) attenuated bone cancer-induced pain hypersensitivities, decreased CX3CR1 expression and glial activation in the spine in a BCP model. Resveratrol (i.t.) also attenuated mechanical allodynia resulting from intrathecally injecting fractalkine in rats. Inhibition of spinal glial activation and CX3CR1 upregulation may involve in resveratrol's analgesic effects. These findings demonstrated that resveratrol attenuated pain facilitation through inhibiting spinal glial activation and CX3CR1 upregulation in a BCP model.

Intrathecal Injection of JWH015 Attenuates Remifentanil-Induced Postoperative Hyperalgesia by Inhibiting Activation of Spinal Glia in a Rat Model

Hyperalgesia and neuroinflammation are associated with glia, which consists of macroglia and microglia. In this study, we used a selective cannabinoid receptor type 2 (CB2) agonist JWH015 to investigate remifentanil-induced postoperative hyperalgesia. Mechanical allodynia and thermal hyperalgesia after postoperative hyperalgesia and intrathecal injection of JWH015 were assessed by the paw withdrawal mechanical threshold and paw withdrawal thermal latency tests. We used immunohistochemistry and immunoblotting to investigate the effect of JWH015 on CB2 receptor, NR2B subunits, activated glial cells, and proinflammatory cytokine expression in rats after remifentanil-induced postoperative hyperalgesia. Postoperative hyperalgesia was induced by intraoperative infusion of remifentanil. Glial cells were activated, and expression levels of several genes were significantly increased, including interleukin 6, tumor necrosis factor α, CB2, and the NR2B subunit phosphorylated at Tyr-1472 (p-NR2B). Intrathecal injection of JWH015 significantly inhibited glial cell activation, suppressed expression of interleukin 6, tumor necrosis factor α, and p-NR2B, and stimulated CB2 expression, thus attenuating postoperative hyperalgesia. However, these phenomena were abolished in the group that was preadministered with AM630. The activation of glia, the production of proinflammatory cytokines, and the expression of CB2 and p-NR2B in the spinal dorsal horn increase significantly during the process of remifentanil-induced hyperalgesia. These changes can be regulated by pretreatment with JWH015, which may be the main mechanism underlying the antihyperalgesia effects of JWH015.

Complete Freund’s adjuvant–induced acute inflammatory pain could be attenuated by triptolide via inhibiting spinal glia activation in rats

BackgroundInflammatory pain is one of the most common clinical symptoms, mechanical allodynia and thermal hypersensitivities are associated with proinflammatory cytokines, and proinflammatory cytokine antagonists could alleviate the hypersensitivity. Previous studies showed that a traditional Chinese medicine ingredient, triptolide could inhibit inflammatory cytokines; however, it was still unknown whether triptolide had beneficial effects on treating inflammatory pain. Materials and methodsThe effects of triptolide on Complete Freund's Adjuvant–induced acute inflammatory pain were investigated using behavioral tests. The activation of spinal glia was morphologically observed by immunofluorescent histochemistry. The levels of OX42, glia fibrillary acidic protein, and phosphorylated extracellular signal–regulated kinase in the spinal cord were detected by Western blot, and the messenger RNA levels of interleukin 1β, interleukin 6, and tumor necrosis factor alpha were detected by real-time polymerase chain reaction. ResultsThese results demonstrate that the triptolide effectively attenuates inflammatory pain induced by Complete Freund's Adjuvant, the underlying mechanism may regulate the phosphorylated extracellular signal–regulated kinase signaling pathway and inhibit the spinal glia activation, and then downregulate the proinflammatory cytokines; the triptolide may be clinically useful as a drug of anti-inflammatory pain. ConclusionsIn the present study, we first reported that repeated systemic administration of triptolide could safely prevent and reverse inflammatory pain. The triptolide may serve as a new potential compound for developing safe therapeutics for patients suffering inflammatory pain.

Induction of Thermal Hyperalgesia and Synaptic Long-Term Potentiation in the Spinal Cord Lamina I by TNF-α and IL-1β is Mediated by Glial Cells

Long-term potentiation (LTP) of synaptic strength in nociceptive pathways is a cellular model of hyperalgesia. The emerging literature suggests a role for cytokines released by spinal glial cells for both LTP and hyperalgesia. However, the underlying mechanisms are still not fully understood. In rat lumbar spinal cord slices, we now demonstrate that conditioning high-frequency stimulation of primary afferents activated spinal microglia within <30 min and spinal astrocytes within ~2 s. Activation of spinal glia was indispensible for LTP induction at C-fiber synapses with spinal lamina I neurons. The cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), which are both released by activated glial cells, were individually sufficient and necessary for LTP induction via redundant pathways. They differentially amplified 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)-propanoic acid receptor-mediated and N-methyl-D-aspartic acid receptor-mediated synaptic currents in lamina I neurons. Unexpectedly, the synaptic effects by IL-1β and TNF-α were not mediated directly via activation of neuronal cytokine receptors, but rather, indirectly via IL-1 receptors and TNF receptors being expressed on glial cells in superficial spinal dorsal horn. Bath application of IL-1β or TNF-α led to the release profiles of pro-inflammatory and anti-inflammatory cytokines, chemokines, and growth factors, which overlapped only partially. Heat hyperalgesia induced by spinal application of either IL-1β or TNF-α in naive animals also required activation of spinal glial cells. These results reveal a novel, decisive role of spinal glial cells for the synaptic effects of IL-1β and TNF-α and for some forms of hyperalgesia.

Differences Between Tumor Necrosis Factor–α Receptors Types 1 and 2 in the Modulation of Spinal Glial Cell Activation and Mechanical Allodynia in a Rat Sciatic Nerve Injury Model

Immunohistological analysis of spinal glial cells and analysis of pain behavior in the rat neuropathic pain model were investigated to clarify the function of tumor necrosis factor (TNF)-α receptors p55 type 1 and p75 type 2. Our objective was to investigate changes in hyperalgesia and glial cell activation after injection of antibodies to each TNF receptor in a rat sciatic nerve injury model. Recent research has revealed that activation of spinal glia plays an important role in radicular and neuropathic pain. TNF-α is reportedly a modulator for glial cell activation; however, the precise relationship between TNF-α and its 2 receptors on glial cells has not been fully delineated. Chronic constriction sciatic nerve injury and sham-operated rats were used. Antibodies to p55 or p75 or saline were intrathecally injected at the L5 level into rats with chronic constriction injury. Mechanical allodynia was examined for 2 weeks. Spinal cords were removed for immunohistochemical studies of ionized calcium-binding adaptor molecule 1 or glial fibrillary acidic protein. Saline rats showed significantly more mechanical allodynia and the number of ionized calcium-binding adaptor molecule 1--immunoreactive microglia and glial fibrillary acidic protein--immunoreactive astrocytes were significantly increased in the saline rats compared with sham-operated rats during the 2 weeks. Injection of both antibodies significantly reduced pain behavior and anti-p55 caused significantly greater reduction compared with anti-p75. The numbers of microglia in both the antibodies groups were significantly decreased when compared with the saline group. In addition, the anti-p55 antibody suppressed microglial activation more than the anti-p75 antibody. These results indicate that the microglial TNF-α p55 pathway played a more important role than the TNF-α p75 pathway in the pathogenesis of peripheral nerve injury pain. This suggests that future studies seeking to clarify neuropathic pain should target TNF-α and p55 receptors in microglia.

Chronic Stress-Induced Changes in Pro-Inflammatory Cytokines and Spinal Glia Markers in the Rat: A Time Course Study

Background/Aims: Spinal glia activation has been proposed as one mechanism underlying visceral hyperalgesia in a rodent model of chronic stress. In order to assess the possible role of changes in circulating cytokines and in blood-spinal cord barrier (BSCB) permeability in spinal glia activation, we studied the time course of peripheral and spinal pro-inflammatory cytokines and of spinal and satellite glia markers in response to repeated water avoidance (WA) stress. Methods: Spinal cords and dorsal root ganglion cells (DRGs) were collected from control rats, rats exposed to 1-hour WA, or 1-hour WA daily for 5 days or 1-hour WA daily for 10 days. Results: We demonstrated a time-dependent change in circulating IL-1β and spinal IL-1β, IL-6 and TNF-α in stressed animals compared with controls. We found altered expression of the astrocyte markers GFAP and Connexin 43 in spinal and DRG samples at different time points. Finally, WA was associated with increased BSCB permeability. Conclusions: These findings confirm the concept that both peripheral and spinal immune markers are altered after chronic WA and suggest a possible link between stress-induced increase of peripheral pro-inflammatory cytokines, changes in satellite glial cells, increase in BSCB permeability and increase in spinal pro-inflammatory mediators suggesting glia activation.

Role of Microglia and Astrocyte in Central Pain Syndrome Following Electrolytic Lesion at the Spinothalamic Tract in Rats

Central pain syndrome (CPS) is a debilitating state and one of the consequences of spinal cord injury in patients. Many pathophysiological aspects of CPS are not well documented. Spinal glia activation has been identified as a key factor in the sensory component of chronic pain. In this study, the role of glial subtypes in the process of CPS induced by unilateral electrolytic lesion of spinothalamic tract (STT) is investigated. Male rats received a laminectomy at T8-T9 and then unilateral electrolytic lesion centered on the STT. Thermal and mechanical thresholds as well as locomotor function were measured on days 0, 3, 7, 14, 21, and 28 post-injuries by tail flick, von Frey filament, and open field tests, respectively. To investigate the spinal glial activation following denervation in STT-lesioned groups, Iba1 and GFAP were detected by immunohistochemistry and Western blotting at the same time points. Data showed that STT lesion significantly decreased thermal pain at day 3 in comparison with sham groups. Significant bilateral allodynia appeared in hind paws at day 14 after spinal cord injury and continued to day 28 (P < 0.05). Additionally, electrolytic spinal lesion attenuated locomotor function of injured animals after 7 days (P < 0.05). In both histological assessments and Western blotting, Iba1 increased at days 3 and 7 while increased GFAP occurred from day 14 to 28 after lesion. It appears that microglial activation is important in the early stages of pain development and astrocytic activation occurs later. These events may lead to behavioral outcomes especially central neuropathic pain.

Activation of astrocytes in the spinal cord contributes to the development of bilateral allodynia after peripheral nerve injury in rats

Activation of spinal cord microglia and astrocytes after peripheral nerve injury contributes to the development of behavioral hypersensitivity. Suppression of spinal cord glial activation attenuates the development of nerve injury-induced allodynia. The contribution of spinal cord glia to existing allodynia, however, is not known. We investigated whether intrathecally administered propentofylline, a glial inhibitor, reverses existing allodynia after nerve injury. Male Sprague–Dawley rats underwent L5 spinal nerve transection, and mechanical allodynia was assessed by measuring hind paw withdrawal thresholds bilaterally using von Frey filaments. Rats received either saline or propentofylline (1, 3, and 10 μg/d) for 7 days (days 0–7) by intrathecal infusion with an osmotic minipump. Other groups of rats received either intrathecal infusion of saline or propentofylline (10 μg/d) for 7 days on days 14–21 or 60–67 after surgery. After completing the intrathecal infusion, lumbar spinal cord sections were assessed for immunostaining of astrocytic glial fibrillary acidic protein and microglial OX-42. Propentofylline infusion on days 0–7 suppressed development of allodynia in both the ipsilateral and contralateral hind paws in a dose-dependent manner. Propentofylline treatment on days 14–21 or 60–67 did not reverse existing allodynia. Propentofylline infusion (10 μg/d) inhibited astrocytic activation bilaterally on days 0–7, 14–21, and 60–67 and inhibited microglial activation on days 14–21 but not on days 0–7 and 60–67. These results suggest that activation of spinal glia, especially astrocytes, dominantly contributes to the development of neuropathic pain and also to mirror-image pain.

Characterization of the acute and persistent pain state present in K/BxN serum transfer arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune arthritis that affects approximately 1% of the population. Synovial inflammation cannot fully explain the level of pain reported by patients and facilitation of pain processing at the spinal level has been implicated. We characterized the K/BxN serum transfer arthritis model as a model of joint inflammation-induced pain and examined pharmacologic responsiveness and spinal glia activation. Mechanical allodynia developed congruently with joint swelling. Surprisingly, allodynia persisted after resolution of inflammation. At the peak of joint inflammation (days 4–10), hypersensitivity was attenuated with i.p. etanercept, gabapentin, and ketorolac. Following resolution of synovial inflammation (days 19–23), only gabapentin relieved allodynia. The superficial dorsal horn of arthritic mice displayed increased staining of microglia at early and late time points, but astrocyte staining increased only during the inflammatory phase. ATF3, a marker of nerve injury, was significantly increased in the lumbar dorsal root ganglia during the late phase (day 28). Hence, serum transfer in the K/BxN serum transfer arthritis model produces a persistent pain state, where the allodynia during the inflammatory state is attenuated by TNF and prostaglandin inhibitors, and the pharmacology and histochemistry data suggest a transition from an inflammatory state to a state that resembles a neuropathic condition over time. Therefore, the K/BxN serum transfer model represents a multifaceted model for studies exploring pain mechanisms in conditions of joint inflammation and may serve as a platform for exploring novel treatment strategies for pain in human arthritic conditions.

Pain Intensity and Duration Can Be Enhanced by Prior Challenge: Initial Evidence Suggestive of a Role of Microglial Priming

Activation of spinal microglia and consequent release of proinflammatory mediators facilitate pain. Under certain conditions, responses of activated microglia can become enhanced. Enhanced microglial production of proinflammatory products may result from priming (sensitization), similar to macrophage priming. We hypothesized that if spinal microglia were primed by an initial inflammatory challenge, subsequent challenges may create enhanced pain. Here, we used a "two-hit" paradigm using 2 successive challenges, which affect overlapping populations of spinal microglia, presented 2 weeks apart. Mechanical allodynia and/or activation of spinal glia were assessed. Initially, laparotomy preceded systemic lipopolysaccharide (LPS). Prior laparotomy caused prolonged microglial (not astrocyte) activation plus enhanced LPS-induced allodynia. In this "two-hit" paradigm, minocycline, a microglial activation inhibitor, significantly reduced later exaggerated pain induced by prior surgery when minocycline was administered intrathecally for 5 days starting either at the time of surgery or 5 days before LPS administration. To test generality of the priming effect, subcutaneous formalin preceded intrathecal HIV-1 gp120, which activates spinal microglia and causes robust allodynia. Prior formalin enhanced intrathecal gp120-induced allodynia, suggesting that microglial priming is not limited to laparotomy and again supporting a spinal site of action. Therefore, spinal microglial priming may increase vulnerability to pain enhancement. Spinal microglia may become "primed" (sensitized) following their activation by disparate forms of peripheral trauma/inflammation. As a result, such primed microglia may overrespond to subsequent challenges, thereby enhancing pain intensity and duration.

S1804 Modulation of Spinal Glia Activation and Glutamate Transmission in the Model of Chronic Water Avoidance Stress-Induced Visceral Hyperalgesia: Role of Glucocorticoids

S1804 Modulation of Spinal Glia Activation and Glutamate Transmission in the Model of Chronic Water Avoidance Stress-Induced Visceral Hyperalgesia: Role of Glucocorticoids

Evoked pain behavior and spinal glia activation is dependent on tumor necrosis factor receptor 1 and 2 in a mouse model of bone cancer pain

Bone–cancer-related pain is one of the most disabling factors in patients suffering from primary bone cancer or bone metastases. Recent studies point toward an important role of proinflammatory cytokines, example tumor necrosis factor-α (TNF), for tumor growth and bone–cancer-associated pain. Mechanisms by which TNF, through its receptor subtypes, TNF receptor 1 (TNFR1) and −2 (TNFR2), elicits altered sensation and pain behavior, are still incompletely understood. To look for a potential role of TNF in bone cancer pain, cancer-related pain was analyzed in fibrosarcoma-bearing C57Bl/6J wild type mice after systemic antagonism of TNF. To further clarify the role of TNF receptor (TNFR) in bone-cancer pain, naive and fibrosarcoma-bearing C57Bl/ 6J wild type and transgenic mice with a deficiency of TNFR1 (TNFR1ko), TNFR2 (TNFR2ko), and TNFR1+2 (TNFR1+2ko) were compared regarding cancer-related pain and hyperalgesia, tumor growth, osteoclast activation, and spinal astrogliosis. Systemic antagonism of TNF significantly alleviated tactile hypersensitivity and spontaneous bone–cancer-related pain behavior. Most interestingly, combined deletion of the TNFR1 and TNFR2, but not of either gene alone, almost completely inhibited the development of tactile hypersensitivity, whereas spontaneous pain behavior was transiently increased. Accordingly, spinal astrogliosis was markedly reduced, whereas tumor growth was significantly increased in TNFR1+2ko mice. In contrast, deletion of the TNFR1 or TNFR2 gene alone did not change tumor growth or spinal astrogliosis. Our findings suggest that the combined absence of TNFR1 and TNFR2 is necessary for the attenuation of cancer-related tactile hypersensitivity and concomitant spinal astrogliosis, whereas tumor growth seems to be inhibited by combined TNFR activation. These findings support the hypothesis of cytokine-dependent pain development in cancer pain. Differential targeting of TNFR activation could be an interesting strategy in bone–cancer-related pain conditions.

Dexmedetomidine blocks thermal hyperalgesia and spinal glial activation in rat model of monoarthritis

To investigate the effect of systemic administration dexmedetomidine, a selective alpha 2 adrenergic receptor (alpha(2)AR) agonist, on thermal hyperalgesia and spinal glial activation evoked by monoarthritis (MA). MA was induced by an intra-articular injection of complete Freund's adjuvant (CFA). Thermal hyperalgesia was measured by Hargreaves' test. The spinal glial activation status was analyzed by GFAP (an astrocytic marker) and Iba-1 (a microglial marker) immunohistochemistry or immunoblotting. Unilateral intra-articular injection of CFA produced a robust glial activation of astrocytes and microglia in the spinal cord, which was associated with the development and maintenance of thermal hyperalgesia. Intraperitoneal (ip) injection of dexmedetomidine (2.5 and 10 microg/kg) was repeatedly given once daily for 5 days with the first injection 60 min before intra-articular CFA. At the dose of 10 microg/kg, dexmedetomidine significantly attenuated MA-induced ipsilateral hyperalgesia from day 2 to day 5. MA-induced up-regulation of GFAP expression on both sides of the spinal dorsal horn was significantly suppressed by day 5 post-MA following dexmedetomidine application, whereas MA-induced Iba-1 up-regulation was only partially suppressed. Systemic dexmedetomidine inhibits the activation of spinal glia, which is possibly associated with its antihyperalgesia in monoarthritic rats.