Characterization of released exosomes from satellite glial cells under normal and inflammatory conditions

Characterization of released exosomes from satellite glial cells under normal and inflammatory... 170Scientific presentations at the 2017 Annual Meeting / Scandinavian Journal of Pain 16 (2017) 165–188(resident) 1 h daily for a week was used. Bodyweight was measuredand blood samples were collected throughout the experiment.Changes in plasma microRNA expression was determined by qPCR.Results: Rats exposed to social stress showed reduced weightgain compared to controls. Preliminary results suggested that socialstress increased the plasma expression of miR-146a-5p, miR-30c5p and miR-223-3p.Conclusions: The data showed that social stress gives reducedweight gain and increased expression of several circulating microRNAs. How this affects the development of persistent pain remainsto be investigated.http://dx.doi.org/10.1016/j.sjpain.2017.04.017Characterization of released exosomes fromsatellite glial cells under normal andinflammatory conditionsM. Duroux ∗ , H.S.H. Vinterhøj, A. Stensballe, P.GazeraniDepartment of Health Science and Technology,Faculty of Medicine, Aalborg University, DenmarkE-mail address: gazerani@hst.aau.dk (P. Gazerani).Aims: Satellite glial cells (SGCs) are non-neuronal cells thatentirely surround neurons within sensory ganglia. This uniquestructure allows SGC-neuron interactions. Altered cross-talk following nerve injury or inflammation is thought to contribute topathogenesis of chronic pain. Release of extracellular vesicles inform of exosomes has been found to play a key role in cell-cellcommunication. However, release of exosomes from SGCs and theirpotential role in modulating pain remain unknown. Hence, thisstudy aimed at identifying and characterizing shed exosomes fromSGCs under normal and inflammatory conditions.Methods: Fresh primary cultures of rat trigeminal ganglia (TG)were prepared from adult male Sprague–Dawley rats. Danish Animal Inspectorate approved the study protocol. Primary SGCs werekept in culture up to 21 days and were characterized by morphologyand immunohistochemistry. Cultured SGCs were monitored undernormal and LPS (50 ng/mL) treatment. Collection of conditionedmedia was performed over time and exosomes were isolated. Particle size distribution and total protein were determined by NTAand LC–MS/MS, respectively.Results: SGCs formed small clusters, spread outwards to areasdevoid of cells but remained spindle-like in appearance with largercell bodies. The primary cultures of SGCs were clearly GS positivewith a low expression of GFAP. LPS treatment led to higher GFAPexpression. Particle size distribution showed that two third of theparticles were in the exosomal size range. Upon LPS-stimulation,four proteins (histone H2B, ubiquitin-60S ribosomal, myosin-9,elongation factor 1-alpha) were found exclusively expressed compared to normal treated SGCs.Conclusions: For the first time it was demonstrated that SGCsshed extracellular vesicles in exosomal size range. Mysoin-9 wasidentified as a possible novel marker of SGCs activation underinflammatory conditions. This protein plays a role in cell-celladhesion and possibly contributes to SGC-SGC cross-talk uponinflammation which may consequently influence the excitabilityof nearby neurons.http://dx.doi.org/10.1016/j.sjpain.2017.04.018Cell-based platform for studying trigeminalsatellite glial cells under normal andinflammatory conditionsH.S.H. Vinterhøj, M. Duroux ∗ , P. GazeraniDepartment of Health Science and Technology,Faculty of Medicine, Aalborg University, DenmarkE-mail address: gazerani@hst.aau.dk (P. Gazerani).Aims: Satellite glial cells (SGCs) in sensory ganglia contribute tothe pathogenesis of chronic pain. In vitro, providing enough freshprimary SGCs poses some practical limitations; hence, frozen stocksof primary cells for culture could be an attractive alternative forcell-based studies or drug screening. This study was designed toinvestigate the morphology and marker expression of frozen andfreshly isolated trigeminal SGCs under normal and inflammatoryconditions.Methods: SGCs from trigeminal ganglia of three maleSprague–Dawley rats and three frozen (sub cultured and passaged)batches of stored primary SGCs were cultured. Their morphologywas observed by phase microscopy and the phenotype was characterized by immunocytochemistry of glutamine synthetase (GS)and glial fibrillary acidic protein (GFAP). Lipopolysaccharide (LPS)was used to simulate a state of neurogenic inflammation in vivo.A pilot test was performed to determine the optimal concentration of LPS to activate SGCs based on GFAP expression. A long-termactivation of the SGCs with 50 ng/mL LPS was chosen for furthercharacterization.Results: The fresh and frozen primary SGCs elicited similarphenotypes based on GS marker expression. However, frozen primary SGCs differed in terms of size and morphology. GFAP wasconstantly expressed in frozen primary SGCs regardless of LPS stimulation. Activation of primary fresh SGCs with LPS spread the GFAPexpression from around the cell body throughout the longer processes and activation was only seen in the LPS treatment.Conclusions: The phenotypic marker, GS was independent ofculture conditions. There was no difference in upregulation of GFAPin thawed SGCs regardless of LPS stimulation. This indicates thatfreeze-thawing might activate SGCs and therefore frozen and passaged cells cannot be suitable for use in cell-based models forinflammation. Fresh primary cells are therefore optimal for studying SGCs under normal and inflammatory conditions.http://dx.doi.org/10.1016/j.sjpain.2017.04.019Tramadol in postoperative pain – 1 mg/ml IVgave no pain reduction but more side effects inthird molar surgeryL. Eriksson a,∗ , L. Sand a,b , T. Gordh a,b,c , Å.Tegelberg a,caUniversity of Uppsala, Uppsala, SwedenUniversity of Oslo, Oslo, Norwayc Malmö University, Malmö, SwedenE-mail address: lars.b.eriksson@ltdalarna.se (L. Eriksson).Aims: Does pre-emptive single dose intravenous tramadol produce a safe and effective postoperative analgesia?Methods: Randomized, placebo controlled, single blindedclinical trial of pre-emptive intravenous tramadol 1 mg/kg in combination with IV midazolam in patients with dental fear. A “Paindiary” evaluates the efficacy. The safety is evaluated perioperativemonitoring (SpO2 and BP).Results: Pain scored by VAS showed no differences between thegroups. It took longer time to first rescue pill in tramadol vs. control group (157 vs. 110 min, p = 0.049). Desaturation (SpO2 < 90%)was more commonly found in tramadol vs. placebo and controlb http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Scandinavian Journal of Pain de Gruyter

Characterization of released exosomes from satellite glial cells under normal and inflammatory conditions

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de Gruyter
Copyright
© 2017 Scandinavian Association for the Study of Pain
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1877-8860
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1877-8879
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10.1016/j.sjpain.2017.04.018
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Abstract

170Scientific presentations at the 2017 Annual Meeting / Scandinavian Journal of Pain 16 (2017) 165–188(resident) 1 h daily for a week was used. Bodyweight was measuredand blood samples were collected throughout the experiment.Changes in plasma microRNA expression was determined by qPCR.Results: Rats exposed to social stress showed reduced weightgain compared to controls. Preliminary results suggested that socialstress increased the plasma expression of miR-146a-5p, miR-30c5p and miR-223-3p.Conclusions: The data showed that social stress gives reducedweight gain and increased expression of several circulating microRNAs. How this affects the development of persistent pain remainsto be investigated.http://dx.doi.org/10.1016/j.sjpain.2017.04.017Characterization of released exosomes fromsatellite glial cells under normal andinflammatory conditionsM. Duroux ∗ , H.S.H. Vinterhøj, A. Stensballe, P.GazeraniDepartment of Health Science and Technology,Faculty of Medicine, Aalborg University, DenmarkE-mail address: gazerani@hst.aau.dk (P. Gazerani).Aims: Satellite glial cells (SGCs) are non-neuronal cells thatentirely surround neurons within sensory ganglia. This uniquestructure allows SGC-neuron interactions. Altered cross-talk following nerve injury or inflammation is thought to contribute topathogenesis of chronic pain. Release of extracellular vesicles inform of exosomes has been found to play a key role in cell-cellcommunication. However, release of exosomes from SGCs and theirpotential role in modulating pain remain unknown. Hence, thisstudy aimed at identifying and characterizing shed exosomes fromSGCs under normal and inflammatory conditions.Methods: Fresh primary cultures of rat trigeminal ganglia (TG)were prepared from adult male Sprague–Dawley rats. Danish Animal Inspectorate approved the study protocol. Primary SGCs werekept in culture up to 21 days and were characterized by morphologyand immunohistochemistry. Cultured SGCs were monitored undernormal and LPS (50 ng/mL) treatment. Collection of conditionedmedia was performed over time and exosomes were isolated. Particle size distribution and total protein were determined by NTAand LC–MS/MS, respectively.Results: SGCs formed small clusters, spread outwards to areasdevoid of cells but remained spindle-like in appearance with largercell bodies. The primary cultures of SGCs were clearly GS positivewith a low expression of GFAP. LPS treatment led to higher GFAPexpression. Particle size distribution showed that two third of theparticles were in the exosomal size range. Upon LPS-stimulation,four proteins (histone H2B, ubiquitin-60S ribosomal, myosin-9,elongation factor 1-alpha) were found exclusively expressed compared to normal treated SGCs.Conclusions: For the first time it was demonstrated that SGCsshed extracellular vesicles in exosomal size range. Mysoin-9 wasidentified as a possible novel marker of SGCs activation underinflammatory conditions. This protein plays a role in cell-celladhesion and possibly contributes to SGC-SGC cross-talk uponinflammation which may consequently influence the excitabilityof nearby neurons.http://dx.doi.org/10.1016/j.sjpain.2017.04.018Cell-based platform for studying trigeminalsatellite glial cells under normal andinflammatory conditionsH.S.H. Vinterhøj, M. Duroux ∗ , P. GazeraniDepartment of Health Science and Technology,Faculty of Medicine, Aalborg University, DenmarkE-mail address: gazerani@hst.aau.dk (P. Gazerani).Aims: Satellite glial cells (SGCs) in sensory ganglia contribute tothe pathogenesis of chronic pain. In vitro, providing enough freshprimary SGCs poses some practical limitations; hence, frozen stocksof primary cells for culture could be an attractive alternative forcell-based studies or drug screening. This study was designed toinvestigate the morphology and marker expression of frozen andfreshly isolated trigeminal SGCs under normal and inflammatoryconditions.Methods: SGCs from trigeminal ganglia of three maleSprague–Dawley rats and three frozen (sub cultured and passaged)batches of stored primary SGCs were cultured. Their morphologywas observed by phase microscopy and the phenotype was characterized by immunocytochemistry of glutamine synthetase (GS)and glial fibrillary acidic protein (GFAP). Lipopolysaccharide (LPS)was used to simulate a state of neurogenic inflammation in vivo.A pilot test was performed to determine the optimal concentration of LPS to activate SGCs based on GFAP expression. A long-termactivation of the SGCs with 50 ng/mL LPS was chosen for furthercharacterization.Results: The fresh and frozen primary SGCs elicited similarphenotypes based on GS marker expression. However, frozen primary SGCs differed in terms of size and morphology. GFAP wasconstantly expressed in frozen primary SGCs regardless of LPS stimulation. Activation of primary fresh SGCs with LPS spread the GFAPexpression from around the cell body throughout the longer processes and activation was only seen in the LPS treatment.Conclusions: The phenotypic marker, GS was independent ofculture conditions. There was no difference in upregulation of GFAPin thawed SGCs regardless of LPS stimulation. This indicates thatfreeze-thawing might activate SGCs and therefore frozen and passaged cells cannot be suitable for use in cell-based models forinflammation. Fresh primary cells are therefore optimal for studying SGCs under normal and inflammatory conditions.http://dx.doi.org/10.1016/j.sjpain.2017.04.019Tramadol in postoperative pain – 1 mg/ml IVgave no pain reduction but more side effects inthird molar surgeryL. Eriksson a,∗ , L. Sand a,b , T. Gordh a,b,c , Å.Tegelberg a,caUniversity of Uppsala, Uppsala, SwedenUniversity of Oslo, Oslo, Norwayc Malmö University, Malmö, SwedenE-mail address: lars.b.eriksson@ltdalarna.se (L. Eriksson).Aims: Does pre-emptive single dose intravenous tramadol produce a safe and effective postoperative analgesia?Methods: Randomized, placebo controlled, single blindedclinical trial of pre-emptive intravenous tramadol 1 mg/kg in combination with IV midazolam in patients with dental fear. A “Paindiary” evaluates the efficacy. The safety is evaluated perioperativemonitoring (SpO2 and BP).Results: Pain scored by VAS showed no differences between thegroups. It took longer time to first rescue pill in tramadol vs. control group (157 vs. 110 min, p = 0.049). Desaturation (SpO2 < 90%)was more commonly found in tramadol vs. placebo and controlb

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Scandinavian Journal of Painde Gruyter

Published: Jul 1, 2017

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