Spinal Cord Glial Cells Research Articles

Contribution of spinal cord glial cells to L. amazonensis experimental infection-induced pain in BALB/c mice

BackgroundThe cellular and molecular pathophysiological mecha\\nisms of pain processing in neglected parasitic infections such as leishmaniasis remain unknown. The present study evaluated the participation of spinal cord glial cells in the pathophysiology of pain induced by Leishmania amazonensis infection in BALB/c mice.MethodsMice received intra-plantar (i.pl.) injection of L. amazonensis (1 × 105) and hyperalgesia, and paw edema were evaluated bilaterally for 40 days. The levels of TNF-α and IL-1β, MPO activity, and histopathology were assessed on the 40th day. ATF3 mRNA expression was assessed in DRG cells at the 30th day post-infection. Blood TNF-α and IL-1β levels and systemic parasite burden were evaluated 5–40 days after the infection. At the 30th day post-infection L. amazonensis, the effects of intrathecal (i.t.) treatments with neutralizing antibody anti-CX3CL1, etanercept (soluble TNFR2 receptor), and interleukin-1 receptor antagonist (IL-1ra) on infection-induced hyperalgesia and paw edema were assessed. In another set of experiments, we performed a time course analysis of spinal cord GFAP and Iba-1 (astrocytes and microglia markers, respectively) and used confocal immunofluorescence and Western blot to confirm the expression at the protein level. Selective astrocyte (α-aminoadipate) and microglia (minocycline) inhibitors were injected i.t. to determine the contribution of these cells to hyperalgesia and paw edema. The effects of i.t. treatments with glial and NFκB (PDTC) inhibitors on spinal glial activation, TNF-α, IL-1β, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation were also evaluated. Finally, the contribution of TNF-α and IL-1β to CX3CL1 mRNA expression was investigated.ResultsL. amazonensis infection induced chronic mechanical and thermal hyperalgesia and paw edema in the infected paw. Mechanical hyperalgesia was also observed in the contralateral paw. TNF-α, IL-1β, MPO activity, and epidermal/dermal thickness increased in the infected paw, which confirmed the peripheral inflammation at the primary foci of this infection. ATF3 mRNA expression at the ipsilateral DRG of the infected paw was unaltered 30 days post-infection. TNF-α and IL-1β blood levels were not changed over the time course of disease, and parasitism increased in a time-dependent manner in the ipsilateral draining lymph node. Treatments targeting CX3CL1, TNF-α, and IL-1β inhibited L. amazonensis-induced ongoing mechanical and thermal hyperalgesia, but not paw edema. A time course of GFAP, Iba-1, and CX3CR1 mRNA expression indicated spinal activation of astrocytes and microglia, which was confirmed at the GFAP and Iba-1 protein level at the peak of mRNA expression (30th day). Selective astrocyte and microglia inhibition diminished infection-induced ipsilateral mechanical hyperalgesia and thermal hyperalgesia, and contralateral mechanical hyperalgesia, but not ipsilateral paw edema. Targeting astrocytes, microglia and NFκB diminished L. amazonensis-induced GFAP, Iba-1, TNF-α, IL-1β, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation in the spinal cord at the peak of spinal cord glial cells activation. CX3CL1 mRNA expression was also detected in the ipsilateral DRG of infected mice at the 30th day post-infection, and the i.t. injection of TNF-α or IL-1β in naïve animals induced CX3CL1 mRNA expression in the spinal cord and ipsilateral DRG.ConclusionsL. amazonensis skin infection produces chronic pain by central mechanisms involving spinal cord astrocytes and microglia-related production of cytokines and chemokines, and NFκB activation contributes to L. amazonensis infection-induced hyperalgesia and neuroinflammation.

The specialised pro-resolving lipid mediator maresin 1 reduces inflammatory pain with a long-lasting analgesic effect.

Maresin 1 (MaR1) is a specialised pro-resolving lipid mediator with anti-inflammatory and analgesic activities. In this study, we addressed the modulation of peripheral and spinal cord cells by MaR1 in the context of inflammatory pain. Mice were treated with MaR1 before intraplantar injection of carrageenan or complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed using the electronic von Frey and thermal hyperalgesia using a hot plate. Spinal cytokine production and NF-κB activation were determined by ELISA and astrocytes and microglia activation by RT-qPCR and immunofluorescence. CGRP release by dorsal root ganglia (DRG) neurons was determined by EIA. Neutrophil and macrophage recruitment were determined by immunofluorescence, flow cytometry, and colorimetric methods. Trpv1 and Nav1.8 expression and calcium imaging of DRG neurons were determined by RT-qPCR and Fluo-4AM respectively. MaR1 reduced carrageenan- and CFA-induced mechanical and thermal hyperalgesia and neutrophil and macrophage recruitment proximal to CGRP+ fibres in the paw skin. Moreover, MaR1 reduced NF-κB activation, IL-1β and TNF-α production, and spinal cord glial cells activation. In the DRG, MaR1 reduced CFA-induced Nav1.8 and Trpv1 mRNA expression and calcium influx and capsaicin-induced release of CGRP by DRG neurons. MaR1 reduced DRG neurons activation and CGRP release explaining, at least in part, its analgesic and anti-inflammatory effects. The enduring analgesic and anti-inflammatory effects and also post-treatment activity of MaR1 suggest that specialised pro-resolving lipid mediators have potential as a new class of drugs for the treatment of inflammatory pain.

Toll-like receptor 2 signaling induces interferon regulatory factor 8 expression in spinal cord microglia after peripheral nerve injury

Although it is well known that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury (PNI), the mechanisms of this activation have not been completely elucidated. We have previously shown that toll-like receptor 2 (TLR2) is involved in PNI-induced spinal cord microglia activation. A more recent study showed that PNI induces interferon regulatory factor 8 (IRF8), which is a key transcription factor responsible for microglia activation. In this study, we investigated the putative role of TLR2 in IRF8 expression in spinal cord microglia after PNI. In TLR2 knockout mice, nerve injury-induced spinal cord IRF8 expression was severely compromised. In addition, an intrathecal injection of lipoteichoic acid (LTA), a TLR2 agonist, induced IRF8 mRNA expression in the spinal cord. Lastly, TLR2 stimulation by LTA induced IRF8 mRNA expression in primary spinal cord glial cells. TLR2-induced IRF8 expression was dependent on p38 MAPK and NF-κB activation. Taken together, these data show that TLR2 activation induces IRF8 expression in spinal cord microglia after PNI.

Tunicamycin inhibits progression of glioma cells through downregulation of the MEG-3-regulated wnt/β-catenin signaling pathway.

Glioma is derived from the oncogenic transformation of brain and spinal cord glial cells, and is one of the most common primary brain tumors. Tunicamycin (TUN) can significantly inhibit glioma growth and aggressiveness by promoting apoptosis in glioma cells. The purpose of the present study was to investigate the effects of TUN on growth of glioma cells and examine the TUN-mediated signaling pathway. The inhibitory effects of TUN on apoptosis, growth, aggressiveness and cell cycle arrest of glioma tumor cells were determined by western blotting, reverse transcription-quantitative polymerase chain reaction, apoptotic assays and immunofluorescence. The results demonstrated that treatment with TUN suppressed growth, migration and invasion of glioma carcinoma cells. In addition, TUN treatment induced apoptosis of glioma cells through downregulation of Bcl-2 and P53 expression levels. Findings also indicated that TUN suppressed proliferation and arrested the glioma cells in the S phase of the cell cycle. Further analysis of the mechanisms of TUN demonstrated that TUN treatment upregulated the expression levels of maternally expressed gene (MEG)-3, wnt and β-catenin in glioma cells. Furthermore, knockdown of MEG-3 expression reversed the TUN-decreased wnt/β-catenin signaling pathway, which subsequently also reversed the TUN-inhibited growth and aggressiveness of glioma cells. In conclusion, the findings in the present study indicated that TUN treatment inhibited growth and aggressiveness through MEG-3-mediated wnt/β-catenin signaling, suggesting that TUN may be an efficient anticancer agent for the treatment of glioma.

Role of Caspase-8 and Fas in Cell Death After Spinal Cord Injury.

Spinal cord injury (SCI) causes the death of neurons and glial cells due to the initial mechanical forces (i.e., primary injury) and through a cascade of secondary molecular events (e.g., inflammation or excitotoxicity) that exacerbate cell death. The loss of neurons and glial cells that are not replaced after the injury is one of the main causes of disability after SCI. Evidence accumulated in last decades has shown that the activation of apoptotic mechanisms is one of the factors causing the death of intrinsic spinal cord (SC) cells following SCI. Although this is not as clear for brain descending neurons, some studies have also shown that apoptosis can be activated in the brain following SCI. There are two main apoptotic pathways, the extrinsic and the intrinsic pathways. Activation of caspase-8 is an important step in the initiation of the extrinsic pathway. Studies in rodents have shown that caspase-8 is activated in SC glial cells and neurons and that the Fas receptor plays a key role in its activation following a traumatic SCI. Recent work in the lamprey model of SCI has also shown the retrograde activation of caspase-8 in brain descending neurons following SCI. Here, we review our current knowledge on the role of caspase-8 and the Fas pathway in cell death following SCI. We also provide a perspective for future work on this process, like the importance of studying the possible contribution of Fas/caspase-8 signaling in the degeneration of brain neurons after SCI in mammals.

PD01-03 STRESS-INDUCED ADRENERGIC DYSREGULATION ALTERS NEURAL-GLIAL DISTRIBUTION AND PHENOTYPE

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis I1 Apr 2017PD01-03 STRESS-INDUCED ADRENERGIC DYSREGULATION ALTERS NEURAL-GLIAL DISTRIBUTION AND PHENOTYPE Bronagh McDonnell, Aura Kullmann, Amanda Wolf-Johnston, Anthony Kanai, Peter Grace, Linda Watkins, Larissa Rodriguez, and Lori Birder Bronagh McDonnellBronagh McDonnell More articles by this author , Aura KullmannAura Kullmann More articles by this author , Amanda Wolf-JohnstonAmanda Wolf-Johnston More articles by this author , Anthony KanaiAnthony Kanai More articles by this author , Peter GracePeter Grace More articles by this author , Linda WatkinsLinda Watkins More articles by this author , Larissa RodriguezLarissa Rodriguez More articles by this author , and Lori BirderLori Birder More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.183AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There is evidence (in patients and preclinical models) that stress can enhance painful sensations in patients with functional pain syndromes such as interstitial cystitis/bladder pain syndrome (IC/BPS). Though the underlying mechanisms have yet to be fully explored, findings reveal increased autonomic (sympathetic) dysregulation as well as a role for central augmentation. In this regard, activation of spinal cord (SC) glial cells can increase excitability of neurons leading to the initiation and maintenance of bladder hyperalgesia and impaired bladder storage function (urgency, frequency). Our goal was to examine whether chronic stress (using the water avoidance stress or WAS model) can alter neural-glial distribution and chemistry, which may play a role in micturition and pain behavior. METHODS Adult female Wistar-Kyoto rats were exposed to WAS by placement on a pedestal in a water-filled container (1hr/day x10 consecutive days) versus handled controls. Previous published findings have revealed WAS rats exhibit long-lasting urinary frequency and hyperalgesia. SC (L6) were harvested from anesthetized animals, and either cryosectioned (for immunocytochemistry) or homogenized (for RT-PCR). The following were investigated: calcitonin gene-related peptide (CGRP; sensory fibers), microglia (IBA-1), Toll-like receptor (TLR-4), purinergic receptor subtypes (P2X4, P2X7). Separate groups of both WAS and control animals were treated 2 days prior then every other day with the adrenergic antagonist phenoxybenzamine (PB; 2 mg/kg i.p.) or saline, respectively. RESULTS WAS increased neural CGRP (40%) and IBA-1 (2 fold) expression in the L6 SC dorsal horn and central canal (regions receiving input from nociceptive fibers). We find PB reduced CGRP expression (92% decrease) as well as IBA-1 in WAS SC. Further, both TLR-4 as well as P2X4 and P2X7 purinergic receptor are increased (50%) in WAS, suggesting microglia activation with chronic stress. CONCLUSIONS Taken together, our findings suggest increased communication between the sympathetic nervous system and bladder sensory neurons that may play an important role in chronic pain conditions. This includes abnormal neural sprouting and altered morphology and chemistry of SC glial cells, which are likely to play an important role in modifying neural activity resulting in changes in bladder function and sensory mechanisms. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e46-e47 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Bronagh McDonnell More articles by this author Aura Kullmann More articles by this author Amanda Wolf-Johnston More articles by this author Anthony Kanai More articles by this author Peter Grace More articles by this author Linda Watkins More articles by this author Larissa Rodriguez More articles by this author Lori Birder More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

(188) Sustained Activation of β2- and β3ARs Leads to Phosphorylation of Neuronal MAPKs and Activation of Glial Cells in Spinal Cord and DRG

(188) Sustained Activation of β2- and β3ARs Leads to Phosphorylation of Neuronal MAPKs and Activation of Glial Cells in Spinal Cord and DRG

An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo

Pathological conditions such as amyotrophic lateral sclerosis, spinal cord injury and chronic pain are characterized by activation of astrocytes and microglia in spinal cord and have been modeled in rodents. In vivo imaging at cellular level in these animal models is limited due to the spinal cord's highly myelinated funiculi. The preparation of acute slices may offer an alternative and valuable strategy to collect structural and functional information in vitro from dorsal, lateral and ventral regions of spinal cord. Here, we describe a procedure for preparing acute slices from mouse spinal cord (Garré et al., 2016). This preparation should allow further understanding of how glial cells in spinal cord respond acutely to various inflammatory challenges.

Prenatal alcohol exposure potentiates chronic neuropathic pain, spinal glial and immune cell activation and alters sciatic nerve and DRG cytokine levels

A growing body of evidence indicates that prenatal alcohol exposure (PAE) may predispose individuals to secondary medical disabilities later in life. Animal models of PAE reveal neuroimmune sequelae such as elevated brain astrocyte and microglial activation with corresponding region-specific changes in immune signaling molecules such as cytokines and chemokines. The aim of this study was to evaluate the effects of moderate PAE on the development and maintenance of allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in adult male rat offspring. Because CCI allodynia requires the actions of glial cytokines, we analyzed lumbar spinal cord glial and immune cell surface markers indicative of their activation levels, as well as sciatic nerve and dorsal root ganglia (DRG) cytokines in PAE offspring in adulthood. While PAE did not alter basal sensory thresholds before or after sham manipulations, PAE significantly potentiated adult onset and maintenance of allodynia. Microscopic analysis revealed exaggerated astrocyte and microglial activation, while flow cytometry data demonstrated increased proportions of immune cells with cell surface major histocompatibility complex II (MHCII) and β-integrin adhesion molecules, which are indicative of PAE-induced immune cell activation. Sciatic nerves from CCI rats revealed that PAE potentiated the proinflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor-alpha (TNFα) protein levels with a simultaneous robust suppression of the anti-inflammatory cytokine, IL-10. A profound reduction in IL-10 expression in the DRG of PAE neuropathic rats was also observed. Taken together, our results provide novel insights into the vulnerability that PAE produces for adult-onset central nervous system (CNS) pathological conditions from peripheral nerve injury.

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco's modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The nitroxyl donor, Angeli’s salt, reduces chronic constriction injury-induced neuropathic pain

Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli’s salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7–14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1β (IL-1β), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1β, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1β and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.

Elevation of Microglial Basic Fibroblast Growth Factor Contributes to Development of Neuropathic Pain after Spinal Nerve Ligation in Rats.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistological analysis of spinal cord and pain behavior analysis in a rat neuropathic pain model were conducted to examine the function of microglial basic fibroblast growth factor (bFGF) in the development of neuropathic pain. To investigate the role of bFGF in spinal microglia during the development of allodynia following spinal nerve ligation in rats. Evidence suggests that the production of bFGF by spinal cord glial cells is increased in response to peripheral nerve injury. Although an association between bFGF and astrocytes has been widely reported, the relationship between bFGF and microglia, particularly with respect to the development of neuropathic pain, remains poorly understood. Spinal nerve ligation rats were used. After surgery, bFGF expression in the spinal cord was investigated using RT-PCR and immunohistochemistry. Neutralizing antibodies to bFGF were injected intrathecally into rats after spinal nerve ligaton. Spinal cords were used for RT-PCR analysis and pain behavior was analyzed using the von Frey test. bFGF mRNA expression was significantly increased in the spinal cord 6 hours after spinal nerve ligation compared with untreated rats. Immunohistochemical analysis revealed that bFGF co-localized with ionized calcium-binding adaptor molecule 1, a microglial marker, and myeloperoxidase. Neutralizing antibodies to bFGF attenuated mechanical allodynia and myeloperoxidase mRNA expression. bFGF increased in spinal microglia during the development allodynia after spinal nerve ligation. Thus, controlling bFGF release from microglia during the acute stage of peripheral nerve injury may suppress the progression of allodynia. N/A.

Glial activation in the periaqueductal gray promotes descending facilitation of neuropathic pain through the p38 MAPK signaling pathway.

The midbrain ventrolateral periaqueductal gray (VL-PAG) is a key component that mediates pain modulation. Although spinal cord glial cells appear to play an important role in chronic pain development, the precise mechanisms involving descending facilitation pathways from the PAG following nerve injury are poorly understood. This study shows that cellular events that occur during glial activation in the VL-PAG may promote descending facilitation from the PAG during neuropathic pain. Chronic constriction nerve injury (CCI) was induced by ligature construction of the sciatic nerve in male Sprague-Dawley rats. Behavioral responses to noxious mechanical (paw withdrawal threshold; PWT) and thermal (paw withdrawal latency; PWL) stimuli were evaluated. After CCI, immunohistochemical and Western blot analysis of microglia and astrocytes in the VL-PAG showed morphological and quantitative changes indicative of activation in microglia and astrocytes. Intra-VL-PAG injection of microglial or astrocytic inhibitors attenuated PWT and PWL at days 7 and 14, respectively, following CCI. We also evaluated the effects of intra-VL-PAG administration of the phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) inhibitor SB 203580 at day 7 after CCI. This treatment abolished microglial activation and produced a significant time-dependent attenuation of PWT and PWL. Western blot analysis showed localized expression of p-p38 in the VL-PAG after CCI. P-p38 was expressed in labeled microglia of the VL-PAG but was not present in astrocytes and neurons on day 7 after CCI. These results demonstrate that CCI-induced neuropathic pain is associated with glial activation in the VL-PAG, which likely participates in descending pain facilitation through the p38 MAPK signaling pathway.

Increased GADD34 in oligodendrocytes in Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid-β (Aβ) and abnormally phosphorylated tau which contribute to endoplasmic reticulum (ER) stress. Previous studies demonstrated that Aβ and a truncated fragment of Aβ induced death of oligodendrocytes in vitro. In addition, a triple-transgenic AD mouse model exhibits significant region-specific alterations in myelination patterns at time points preceding the appearance of Aβ accumulation. The growth arrest and DNA damage protein (GADD) 34 is up-regulated in response to ER stress and regulates subunit of protein phosphatase 1 (PP1) complex that dephosphorylates eukaryotic translation initiator factor 2α (elF2α). Thus, GADD34 is known as an ER stress regulator or ER stress marker. In a recent study, GADD34 was induced in the spinal cord glial cells of an amyotrophic lateral sclerosis (ALS) mouse model. It is interesting that reduced GADD34 delayed the onset of ALS and prolonged the survival period in the mouse model. In this study, we have demonstrated that GADD34 was increased in neurons of human AD brains. Additionally, this finding was also observed in oligodendrocytes in human AD brains. Furthermore, we showed that the expression levels of GADD34 in neurons and oligodendrocytes were significantly increased in the early stage of AD in the mouse model. As oligodendrocytes were more affected in the early stages of AD in this experimental model, ER stress of Aβ oligomers may be more related to oligodendrocytes than to neurons. These results suggest that GADD34 could be a therapeutic target for preventing ER stress in neuronal cells in AD.

Intrathecal injection of fluorocitric acid inhibits the activation of glial cells causing reduced mirror pain in rats.

BackgroundGrowing evidence has shown that unilateral nerve injury results in pain hypersensitivity in the ipsilateral and contralateral sides respective to the injury site. This phenomenon is known as mirror image pain (MIP). Glial cells have been indicated in the mechanism of MIP; however, it is not clear how glial cells are involved in MIP.MethodsTo observe phenomenon MIP and the following mechanism, 20 adult male Sprague–Dawley rats (weighing 180–220 g) were separated into two groups: Sham Group (n = 10) and left L5 spinal nerve ligated and sectioned (SNL) group (n = 10). Thermal hyperalgesia and mechanical hypersensitivity were measured for both groups to determine if the SNL model had Mirror image of Pain (MIP). Nav1.7 protein expression in DRG was analyzed using immunohistochemistry and western-blotting. And then to observe the effect of fluorocitrate on MIP, 15 rats were separated into three Groups: Sham Group (n = 5); SNL + FC group: intrathecal injection of Fluorocitric acid(FC) 1 nmol/10 μL (n = 5); SNL + NS group: intrathecal injection of 0.9% Normal Saline (n = 5). Behavior testing, immunocytochemistry, and western-blotting using dorsal root ganglion (DRG) from both sides were then conducted.ResultsThe results showed pain hypersensitivity in both hind-paws of the SNL animals, Mechanical tests showed the paw withdrawal threshold dropped from 13.30 ± 1.204 g to 2.57 ± 1.963 g at 14 d as will as the ipsilateral paw thermal withdrawal threshold dropped from 16.5 ± 2.236 s to 4.38 ± 2.544 s at 14d. Mechanical tests showed the contralateral paw withdrawal threshold dropped from 14.01 ± 1.412 to 4.2 ± 1.789 g at 7d will the thermal withdrawal threshold dropped from 16.8 ± 2.176 s to 7.6 ± 1.517 s at 7d. Nav1.7 expression increased and glial cells actived in bilateral side DRG after SNL compared with sham group. After intrathecal injection of fluorocitrate, the glial cell in bilatral DRG were inhibited and the pain behavior were reversed in both hindpaws too.ConclusionsFluorocitrate can inhibit the activation of glial cells in spinal cord and DRG, and reduce MIP.

Characterization of spinal cord glial cells in a model of hindlimb unloading in mice

Exposure to microgravity has been shown to result in damaging alterations to skeletal muscle, bones, and inner organs. In this study, we investigated the effects of microgravity by using a hindlimb unloading model (HUM) in mice. The characteristics of the lumbar spinal cords of HUM mice 30days after hindlimb unloading were examined. Morphometric analysis showed reductions of the total area, gray matter, and white matter by 17%, 20%, and 12%, respectively. Myelinated fibers in the white matter showed prominent myelin destruction. Analysis of the number of glial fibrillary acidic protein (GFAP+)/S100 calcium-binding protein B (S100B−), GFAP+/S100B+, and GFAP−/S100B+ astrocytes in the ventral horn (VH), central channel area (CC), dorsal root entry zone (DREZ), main corticospinal tract (CST), and ventral funiculi (VF) showed that the number of GFAP+/S100B− astrocytes was increased in the DREZ and CST of HUM mice. Additionally, GFAP+/S100B+ cell numbers were significantly decreased in the VH and CST but did not differ in the CC or DREZ of HUM mice, as compared with the control. The numbers of GFAP−/S100B+ cells were significantly reduced only in the VH of HUM mice. Moreover, the number of ionized calcium-binding adaptor molecule 1 (Iba1+) microglia cells was significantly increased in the CC and DREZ of HUM mice. In control mice, homeobox protein HoxB8 (HoxB8+) cells were found only in the CC; in contrast, HoxB8+ cells were observed in all studied areas in HUM mice, with the greatest number found in the CC. Genome-wide transcriptome analysis of the lumbar spinal cords of HUM mice showed decreased expression of genes encoding myelin, extracellular matrix, cytoskeleton, and cell adhesion proteins. Real-time polymerase chain reaction (PCR) confirmed reductions in the expression of mpz, pmp2, pmp22, and prx genes, which are involved in myelination, as well as decreases in the levels of genes encoding extracellular matrix molecules, including glycoproteins (matrix gla protein (MGP), osteoglycin (OGN), microfibrillar associated protein 5 (MFAP), and collagen, type IV, alpha 1 (COL4A)), proteoglycans (perlecan (heparan sulfate proteoglycan) (HSPG)), and metalloproteinases (lysyl oxidase (LOX)). Thus, our results showed that hindlimb unloading caused decreases in gray and white matter areas, changes in gene expression, alterations in myelination, and phenotypic modifications in glial cells in the lumbar spinal cords of mice.

Neurotrophic factor derived from spinal cord glial cell line in rats with neuropathic pain involved in the modulation of dopamine D2 receptor agonists hyperalgesia

Objective To study the effects of intrathecal injection of dopamine D2 receptor agonist quinpirole on expression of glial cell line-derived neurotrophic factor in the spinal cord in rats with chronic constrictive injury (CCI) and explore its possible mechanism of mediating antinociception.Methods In this study,models were established CCI in male Sprague-Dawley rats.The experiment was performed as 2 parts.In part one,30 CCI rats were randomly divided into 5 groups (n=6):CCI+saline (group NS),CCI+quinpirole 0.1 μg (group Q0.1),CCI+quinpirole 1 μg (group Q1),CCI+quinpirole 5 μg (group Q5),CCI+quinpirole 10μg (group Q 10).The drugs were injected intrathecally on day 7 after CCI,respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured before and at 0.5,1,2,4,8 h and 16 h after intrathecal injection.In part two 54 CCI rats were randomly divided into 3 groups (group Q5,Q10 and NS,n=18).Group Q5 and Q10 were sacrificed at 0.5,1,2,4,8,16 h after intrathecal quinpirole 5,10 μg on day 7 after CCI.Group NS,a single intrathecal injection of saline 10 μl.Group M,another 6 normal rats as model control group.Another 6 normal rats as control group C.The expression of glial cell linederived neurotrophic fact (GDNF) in the spinal cord was determined by Western blot.Results Part one:compared with group NS,the PWMT and PWTL of group Q0.1 at each time point after injection had no statistically significant difference (P＞0.05).The PWMT and PWTL of 2 h of group Q1 [(4.3±1.5) g,(13.2±1.6) s].1,2,4 h of group Q5 [(4.7±1.6),(5.3±1.6),(4.7±2.1) g,(14.0±1.7),(15.2±1.5),(13.4±1.6) s] and Q10 [(6.0±1.3),(7.3±1.0),(5.3±2.1) g,(15.3±1.8),(17.5±1.2),(14.9±1.7) s] after drug administration were significantly promoted (P＜0.05).Part two:compared with group C,the expression of GDNF was significantly increased in group M (0.95±0.09)(P＜0.05).Compared with group M and NS,group Q5 [(1.47±0.12),(1.24±0.05)] and Q10[(1.63±0.08),(1.27±0.06)] significantly increased the expression of GDNF (P＜0.05),and it can last until 4 h after injection at least.Conclusions Intrathecal injection of quinpirole can significantly increase the level of GDNF expression in the spinal cord.At the same time,the pain behavior was improved.The upregulation of GNDF expression is involved in the modulation of dopamine D2 receptor agonist on neuropathic pain. Key words: Neuropathic pain; Dopamine agonist; Spinal; Glial cell line-derived neurotrophic fact; Intrathecal injection

G protein-coupled receptor, family C, group 5 (GPRC5B) downregulation in spinal cord neurons is involved in neuropathic pain.

BackgroundG protein-coupled receptor, family C, group 5 (GPRC5B), a retinoic acid-inducible orphan G-protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins presumably related in non-canonical Wnt signaling. In this study, we investigated altered GPRC5B expression in the dorsal horn of the spinal cord after spinal nerve injury and its involvement in the development of neuropathic pain.MethodsAfter induction of anesthesia by intraperitoneal injection of pentobarbital (35 mg /kg), the left L5 spinal nerve at the level of 2 mm distal to the L5 DRG was tightly ligated with silk and cut just distal to the ligature. Seven days after nerve injury, animals were perfused with 4% paraformaldehyde, and the spinal cords were extracted and post-fixed at 4℃ overnight. To identify the expression of GPRC5B and analyze the involvement of GPRC5B in neuropathic pain, immunofluorescence was performed using several markers for neurons and glial cells in spinal cord tissue.ResultsAfter L5 spinal nerve ligation (SNL), the expression of GPRC5B was decreased in the ipsilateral part, as compared to the contralateral part, of the spinal dorsal horn. SNL induced the downregulation of GPRC5B in NeuN-positive neurons in the spinal dorsal horn. However, CNPase-positive oligodendrocytes, OX42-positive microglia, and GFAP-positive astrocytes were not immunolabeled with GPRC5B antibody in the spinal dorsal horn.ConclusionsThese results imply that L5 SNL-induced GPRC5B downregulation may affect microglial activation in the spinal dorsal horn and be involved in neuropathic pain.

Improved axonal regeneration after spinal cord injury in mice with conditional deletion of ephrin B2 under the GFAP promoter

Spinal cord injury (SCI) initiates a cascade of processes that ultimately form a nonpermissive environment for axonal regeneration. Emerging evidence suggests that regenerative failure may be due in part to inhibitory factors expressed by reactive spinal cord glial cells and meningeal fibroblasts, such as the Eph receptor protein-tyrosine kinases and their corresponding ligands (ephrins). Here we sought to assess the role of ephrin B2, an inhibitory axonal guidance molecule, as an inhibitor of the recovery process following SCI. To determine the extent of ephrin B2 involvement in axonal regenerative failure, a SCI model was performed on a conditional ephrin B2 knockout mouse strain (ephrin B2−/−), in which the ephrin B2 gene was deleted under the GFAP promoter . The expression of ephrin B2 was significantly decreased in astrocytes of injured and uninjured ephrin B2−/− mice compared to wild-type mice. Notably, in the ephrin B2−/− mice, the deletion of ephrin B2 reduced astrogliosis, and accelerated motor function recovery after SCI. Anterograde axonal tracing on a hemisection model of SCI further showed that ephrin B2−/− mice exhibited increased regeneration of injured corticospinal axons and a reduced glial scar, when compared to littermate controls exposed to similar injury. These results were confirmed by an in vitro neurite outgrowth assay and ephrin B2 functional blockage, which showed that ephrin B2 expressed on astrocytes inhibited axonal growth. Combined these findings suggest that ephrin B2 ligands expressed by reactive astrocytes impede the recovery process following SCI.

Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells in vitro. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.