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T108 PAIN SENSITIVITY AND PERIPHERAL INFLAMMATION ARE INCREASED IN A RAT MODEL OF GENETIC OBESITY
POSTER SESSIONS / European Journal of Pain Supplements 5 (2011) 15–295
T107 NEURITES OF PORCINE DRG NEURONS DIFFERENTIALLY SUPPORTED BY TROPHIC FACTORS AND CO-CULTURED WITH KERATINOCYTES A. Klusch1 , L. Ponce2 , M. Ringkamp3 , I. Schafer ¨ 2 , A. Holloschi2 , 1 2 1 1 M. Schmelz , M. Hafner , M. Petersen *. Anesthesiology, Universit¨ atsmedizin Mannheim, 2 Inst. Molecular Cell Biol., Hochschule Mannheim, Mannheim, Germany; 3 Neurosurgery, Johns Hopkins University, Baltimore, MD, USA Background and Aims: Our long term goal is to study the role of non-neuronal cells like keratinocytes in nerve terminal excitability. Methods: Outgrown neurites from dissociated porcine DRG neurons and porcine epidermal keratinocytes were used as a model. Neurites were spatially separated from their somata in a compartmented chamber. Somata were cultured in the central compartment. Outgrowth of neurites into the side compartments was supported by trophic factors NGF, GDNF or artemin. One side compartment contained medium without growth factors, the other one NGF, GDNF or artemin in different concentrations. For co-culture, keratinocytes were seeded in the side compartments in keratinocyte medium. Ca2+ -imaging was performed to detect responses in neurites and keratinocytes. Results: In mono-culture, with NGF in the central compartment, processes grew only into the growth factor containing side compartment. With GDNF or artemin, there was neurite outgrowth also into the control side. With NGF or GDNF in the lateral compartment, about 60% of the neurites were capsaicin responsive. With GDNF in the central, but not in the lateral compartment, a clearly lower proportion was responsive. To investigate neurite/ keratinocyte interaction, in a first step, we established a co-culture. Neurites only were activated by a cross-compartment activation through KCl applied to the somata-containing compartment. This stimulation revealed a change in cytosolic calcium concentration in neurites and in adjacent keratinocytes. Conclusions: Using a compartmented chamber, neurites of nociceptive neurons can be utilized to investigate information processing alone or in co-culture with non-neuronal cells. Support: Land Baden-Wurttemberg ¨ and DFG, KFG 107. Disclosure: None declared
T108 PAIN SENSITIVITY AND PERIPHERAL INFLAMMATION ARE INCREASED IN A RAT MODEL OF GENETIC OBESITY T. Iannitti *, A. Graham, S. Dolan. Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK Background and Aims: Obese individuals are more likely to be affected by chronic pain conditions such as osteoarthritis, migraine and musculoskeletal conditions, and alterations in pain sensitivity have been reported in obese rats, however the mechanisms underlying these changes are not known. The aim of this study was to investigate whether acute nociception and inflammatory hyperalgesia are altered in a rodent model of genetic obesity. Methods: Hindpaw withdrawal latency to thermal stimulation, response thresholds to mechanical stimulation, and paw oedema were assessed in male Zucker fatty rats (fa/fa) and their lean littermates (fa/−) (n = 6–9 per group) in the absence of inflammation, then in response to intradermal hindpaw injection of carrageenan (3%; 50 ml) or capsaicin (10 mg; 50 ml) or hindpaw incision. Results: There was no difference in acute thermal or mechanical nociceptive responses between obese or lean animals. Following carrageenan-induced inflammation, obese rats were significantly more sensitive to mechanical and thermal stimulation of the inflamed paw, and displayed greater paw oedema compared to lean rats. No difference in capsaicin – or paw incision induced – mechanical and thermal hyperalgesia were observed between obese and lean rats.
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Conclusions: The increased hyperalgesia and peripheral inflammation in obese rats fits well with the hypothesis that obesity is a chronic low-grade inflammatory disorder, producing a state where responses to subsequent inflammatory challenge are potentiated. These alterations may underlie the increased susceptibilty of obese individuals to develop chronic inflammatory pain conditions, and signal the need for more aggressive analgesic treatment regimes in these individuals. Disclosure: None declared
T109 THE ROLE OF THE LYSOPHOSHATIDYLINOSITOL–GPR55 PATHWAY IN PERIPHERAL SENSITIZATION IN PAIN STATES V. Gangadharan *, M. Kurejova, D. Skoricova, C. Njoo, D. Selvaraj, R. Kuner. Institute of Pharmacology, Heidelberg University, Heidelberg, Germany Background and Aims: Recent studies have suggested that the orphan receptor GPR55 is activated by multiple different cannabinoid ligands and also by L-a-lysophosphatidylinositol (LPI). GPR55 is reported to be expressed on sensory neurons and mice lacking GPR55 were shown to have reduced mechanical hypersensitivity in experimental pain models, suggesting a pronociceptive role of GPR55. LPI was found to be secreted in large quantities from varieties of cancer tissue. These facts led us to speculate that LPI released from cancer cells could sensitize the nociceptors and thereby lead to cancer-induced pain. The aim of the study was to investigate the role of LPI in cancer induced, mechanically evoked pain and its possible underlying mechanism. Methods: Mice were treated with intraplantar injections of either LPI or vehicle and mechanical sensitivity was measured using Von Frey filaments. Inflammatory and demyelinating capacity of LPI were studied using immunohistochemistry and toluidine blue staining/electron microscopy. Dorsal root ganglion were cultured and used for western blot analysis. Results: We found that the dorsal root ganglion neurons were directly activated by LPI in vitro. We also found that intraplantar injection of LPI lead to a mechanical hypersensitivity in a dose dependent manner in wild type mice, without eliciting any inflammation or demyelination. LPI induced mechanical hypersensitivity was significantly reduced in mice lacking GPR55. Conclusions: These results indicate a possible role of LPI/GPR55 signaling pathway in cancer induced pain. Blocking of GPR55 signaling with specific ligands might therefore serve as a potential therapeutic strategy to treat cancer induced pain. Disclosure: None declared
T110 CRITICAL RESIDUES OF THE INTRACELLULAR C-TERMINAL DOMAIN OF ATP-GATED P2X3 RECEPTORS REGULATE FUNCTIONAL DIFFERENCES BETWEEN HUMAN AND RAT RECEPTORS M. Sundukova1 *, S. Vilotti1 , N. Kavˇciˇc2 , R. Abbate1 , E. Fabbretti1,2 , A. Nistri1 . 1 Neurobiology Sector and IIT Unit, SISSA, Trieste, Italy; 2 University of Nova Gorica, Nova Gorica, Slovenia Background and Aims: ATP-activated P2X3 receptors of sensory neurons contribute to pain transduction and are involved in the pathogenesis of chronic pain. The intracellular kinase Csk phosphorylates tyrosine 393 on the C-terminal of rat P2X3 receptor, and inhibits its function. Although rat and human P2X3 receptors are rather homologous, in human P2X3 receptors tyrosine 393 is replaced by phenylalanine. In this study we characterized point mutations of key amino acid residues in the C-terminals of the human and rat receptors, to better understand the molecular requirements for their function. Methods: P2X3 receptor-mediated currents were recorded with whole cell patch clamping from HEK 293T cells transiently expressing human or rat wild-type P2X3 receptors, or selected mutants in C-terminal domain. Constructs have been sequenced
T107 NEURITES OF PORCINE DRG NEURONS DIFFERENTIALLY SUPPORTED BY TROPHIC FACTORS AND CO-CULTURED WITH KERATINOCYTES A. Klusch1 , L. Ponce2 , M. Ringkamp3 , I. Schafer ¨ 2 , A. Holloschi2 , 1 2 1 1 M. Schmelz , M. Hafner , M. Petersen *. Anesthesiology, Universit¨ atsmedizin Mannheim, 2 Inst. Molecular Cell Biol., Hochschule Mannheim, Mannheim, Germany; 3 Neurosurgery, Johns Hopkins University, Baltimore, MD, USA Background and Aims: Our long term goal is to study the role of non-neuronal cells like keratinocytes in nerve terminal excitability. Methods: Outgrown neurites from dissociated porcine DRG neurons and porcine epidermal keratinocytes were used as a model. Neurites were spatially separated from their somata in a compartmented chamber. Somata were cultured in the central compartment. Outgrowth of neurites into the side compartments was supported by trophic factors NGF, GDNF or artemin. One side compartment contained medium without growth factors, the other one NGF, GDNF or artemin in different concentrations. For co-culture, keratinocytes were seeded in the side compartments in keratinocyte medium. Ca2+ -imaging was performed to detect responses in neurites and keratinocytes. Results: In mono-culture, with NGF in the central compartment, processes grew only into the growth factor containing side compartment. With GDNF or artemin, there was neurite outgrowth also into the control side. With NGF or GDNF in the lateral compartment, about 60% of the neurites were capsaicin responsive. With GDNF in the central, but not in the lateral compartment, a clearly lower proportion was responsive. To investigate neurite/ keratinocyte interaction, in a first step, we established a co-culture. Neurites only were activated by a cross-compartment activation through KCl applied to the somata-containing compartment. This stimulation revealed a change in cytosolic calcium concentration in neurites and in adjacent keratinocytes. Conclusions: Using a compartmented chamber, neurites of nociceptive neurons can be utilized to investigate information processing alone or in co-culture with non-neuronal cells. Support: Land Baden-Wurttemberg ¨ and DFG, KFG 107. Disclosure: None declared
T108 PAIN SENSITIVITY AND PERIPHERAL INFLAMMATION ARE INCREASED IN A RAT MODEL OF GENETIC OBESITY T. Iannitti *, A. Graham, S. Dolan. Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK Background and Aims: Obese individuals are more likely to be affected by chronic pain conditions such as osteoarthritis, migraine and musculoskeletal conditions, and alterations in pain sensitivity have been reported in obese rats, however the mechanisms underlying these changes are not known. The aim of this study was to investigate whether acute nociception and inflammatory hyperalgesia are altered in a rodent model of genetic obesity. Methods: Hindpaw withdrawal latency to thermal stimulation, response thresholds to mechanical stimulation, and paw oedema were assessed in male Zucker fatty rats (fa/fa) and their lean littermates (fa/−) (n = 6–9 per group) in the absence of inflammation, then in response to intradermal hindpaw injection of carrageenan (3%; 50 ml) or capsaicin (10 mg; 50 ml) or hindpaw incision. Results: There was no difference in acute thermal or mechanical nociceptive responses between obese or lean animals. Following carrageenan-induced inflammation, obese rats were significantly more sensitive to mechanical and thermal stimulation of the inflamed paw, and displayed greater paw oedema compared to lean rats. No difference in capsaicin – or paw incision induced – mechanical and thermal hyperalgesia were observed between obese and lean rats.
17
Conclusions: The increased hyperalgesia and peripheral inflammation in obese rats fits well with the hypothesis that obesity is a chronic low-grade inflammatory disorder, producing a state where responses to subsequent inflammatory challenge are potentiated. These alterations may underlie the increased susceptibilty of obese individuals to develop chronic inflammatory pain conditions, and signal the need for more aggressive analgesic treatment regimes in these individuals. Disclosure: None declared
T109 THE ROLE OF THE LYSOPHOSHATIDYLINOSITOL–GPR55 PATHWAY IN PERIPHERAL SENSITIZATION IN PAIN STATES V. Gangadharan *, M. Kurejova, D. Skoricova, C. Njoo, D. Selvaraj, R. Kuner. Institute of Pharmacology, Heidelberg University, Heidelberg, Germany Background and Aims: Recent studies have suggested that the orphan receptor GPR55 is activated by multiple different cannabinoid ligands and also by L-a-lysophosphatidylinositol (LPI). GPR55 is reported to be expressed on sensory neurons and mice lacking GPR55 were shown to have reduced mechanical hypersensitivity in experimental pain models, suggesting a pronociceptive role of GPR55. LPI was found to be secreted in large quantities from varieties of cancer tissue. These facts led us to speculate that LPI released from cancer cells could sensitize the nociceptors and thereby lead to cancer-induced pain. The aim of the study was to investigate the role of LPI in cancer induced, mechanically evoked pain and its possible underlying mechanism. Methods: Mice were treated with intraplantar injections of either LPI or vehicle and mechanical sensitivity was measured using Von Frey filaments. Inflammatory and demyelinating capacity of LPI were studied using immunohistochemistry and toluidine blue staining/electron microscopy. Dorsal root ganglion were cultured and used for western blot analysis. Results: We found that the dorsal root ganglion neurons were directly activated by LPI in vitro. We also found that intraplantar injection of LPI lead to a mechanical hypersensitivity in a dose dependent manner in wild type mice, without eliciting any inflammation or demyelination. LPI induced mechanical hypersensitivity was significantly reduced in mice lacking GPR55. Conclusions: These results indicate a possible role of LPI/GPR55 signaling pathway in cancer induced pain. Blocking of GPR55 signaling with specific ligands might therefore serve as a potential therapeutic strategy to treat cancer induced pain. Disclosure: None declared
T110 CRITICAL RESIDUES OF THE INTRACELLULAR C-TERMINAL DOMAIN OF ATP-GATED P2X3 RECEPTORS REGULATE FUNCTIONAL DIFFERENCES BETWEEN HUMAN AND RAT RECEPTORS M. Sundukova1 *, S. Vilotti1 , N. Kavˇciˇc2 , R. Abbate1 , E. Fabbretti1,2 , A. Nistri1 . 1 Neurobiology Sector and IIT Unit, SISSA, Trieste, Italy; 2 University of Nova Gorica, Nova Gorica, Slovenia Background and Aims: ATP-activated P2X3 receptors of sensory neurons contribute to pain transduction and are involved in the pathogenesis of chronic pain. The intracellular kinase Csk phosphorylates tyrosine 393 on the C-terminal of rat P2X3 receptor, and inhibits its function. Although rat and human P2X3 receptors are rather homologous, in human P2X3 receptors tyrosine 393 is replaced by phenylalanine. In this study we characterized point mutations of key amino acid residues in the C-terminals of the human and rat receptors, to better understand the molecular requirements for their function. Methods: P2X3 receptor-mediated currents were recorded with whole cell patch clamping from HEK 293T cells transiently expressing human or rat wild-type P2X3 receptors, or selected mutants in C-terminal domain. Constructs have been sequenced