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Megumu Yoshimura - One of the best experts on this subject based on the ideXlab platform.

  • differential effects of alpha 1 adrenoceptor antagonists on the postsynaptic sensitivity using Slice patch clamp technique for inhibitory postsynaptic current in substantia gelatinosa neurons from lumbosacral Spinal Cord in rats
    International Neurourology Journal, 2020
    Co-Authors: Daisuke Uta, Megumu Yoshimura, Tsuyoshi Hattori
    Abstract:

    Purpose Alpha1-adrenoceptors participate in improving storage symptoms of male lower urinary tract symptoms. However, the mechanism of action of these compounds remains unclear. The goal of the present study was to clarify the effect of α1- adrenoceptor antagonists on γ-aminobutyric acid (GABA)/glycine-mediated outward currents of the inhibitory postsynaptic current (IPSC) in substantia gelatinosa (SG) neurons from the lumbosacral Spinal Cord in rats. Methods Male adult Sprague-Dawley rats were used. Blind whole-cell patch-clamp reCordings were performed in SG neurons from isolated Spinal Cord Slice preparations. IPSCs were reCorded in individual SG neurons to which naftopidil (100μM), tamsulosin (100μM), silodosin (30μM), or prazosin (10μM) were applied sequentially with intervening washout periods. Strychnine (2μM), bicuculline (10μM), or tetrodotoxin (TTX)(1μM) were added before naftopidil. Individual outward currents were analyzed. Results The bath application of naftopidil, yielded outward IPSCs in 13 of 52 SG neurons. The naftopidil response was unchanged in the presence of TTX. Regression analysis of the outward currents between the 1st and 2nd applications of naftopidil revealed a Pearson correlation coefficient of 0.996 with a line slope of 0.983. The naftopidil-induced outward current was attenuated in the presence of strychnine and/or bicuculline. The GABA/glycine-mediated outward currents induced by tamsulosin, silodosin, and prazosin were smaller than those obtained with naftopidil. Conclusion Naftopidil-induced GABA/glycine-mediated outward currents in a subset of SG neurons prepared from the L6- S1 level of rat Spinal Cord. The results indicated that α1-adrenoceptor antagonists, particularly naftopidil, induce neural suppression (in part) by mediating hyperpolarization. The response is associated with glycinergic and/or GABAergic neural transmission. Naftopidil may suppress the micturition reflex and improve urinary storage symptoms as a subsidiary effect resulting from hyperpolarization in SG neurons of the Spinal Cord.

  • effect of alpha 1 adrnoceptor antagonists on postsynaptic sensitivity in substantia gelatinosa neurons from lumbosacral Spinal Cord in rats using Slice patch clamp technique for mepsc
    International Neurourology Journal, 2020
    Co-Authors: Daisuke Uta, Megumu Yoshimura, Tsuyoshi Hattori
    Abstract:

    Purpose Alpha1-adrenoceptors participate in improving storage symptoms of male lower urinary tract symptoms (LUTS). However, the mechanism of action of these compounds remains unclear. To clarify the mechanism of the α1-adrenoceptor antagonists, the amplitude of miniature excitatory postsynaptic currents (mEPSCs) was analyzed in the lumbosacral Spinal Cord in rats. Methods Male adult Sprague-Dawley rats were used. Blind whole-cell patch-clamp reCordings were performed on substantia gelatinosa (SG) neurons in Spinal Cord Slice preparations. The amplitude of mEPSCs was reCorded in individual SG neurons to which α1-adrenoceptors (100μM naftopidil, 100μM tamsulosin, and 30μM silodosin) were applied sequentially with intervening washout periods. Individual amplitudes were analyzed. Results Pearson correlation coefficients (r) for the amplitudes of mEPSCs between the baseline and postadministration of α1- adrenoceptor antagonists indicated changes of the amplitude ranked in the order of naftopidil (r =0.393), tamsulosin (r=0.738), and silodosin (r=0.944). Together, the α1-adrenoceptor antagonists yielded significant increases in the amplitude of mEPSCs in SG neurons (n=108, P=0.012). However, the effects of each α1-adrenoceptor antagonist on the amplitude were as follows (relative to the baseline; n=36 each): naftopidil, P=0.129; tamsulosin, P=0.201; and silodosin, P=0.005. The rate of response to naftopidil for the outward current was relatively high among the α1-adrenoceptor blockers. An inward current was observed only with the naftopidil application. Conclusion Alpha1-adrenoceptor antagonists changed the amplitudes of mEPSCs in a subset of SG neurons in Slices prepared from the L6-S1 levels of rat spine. Although the α1-adrenoceptor antagonists generated inward or outward currents in the SG neurons, different rates of response were observed with each antagonist. These results are important for understanding the mechanisms of action (at the Spinal level) of α1-adrenoceptor antagonists for the storage symptoms of male LUTS.

  • actions of propofol on substantia gelatinosa neurones in rat Spinal Cord revealed by in vitro and in vivo patch clamp reCordings
    European Journal of Neuroscience, 2009
    Co-Authors: Hidemasa Furue, Tomonori Takazawa, Koichi Nishikawa, Daisuke Uta, Kaori Takeshima, Fumio Goto, Megumu Yoshimura
    Abstract:

    Propofol, an intravenous general anaesthetic, exerts anaesthetic actions through interaction with gamma-aminobutyric acid type A (GABA(A)) receptors in the supraSpinal nervous system. However, whether propofol has any significant effects on synaptic transmission at the Spinal level and whether it exhibits antinociceptive action is still not fully clarified. Spontaneous activity and stimulus-evoked responses of substantia gelatinosa (SG) neurones to noxious pinch stimuli were reCorded using spontaneously breathing rats under propofol anaesthesia using in vivo whole-cell patch-clamp techniques. Precise actions of propofol on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) as well as excitatory postsynaptic currents (EPSCs) in SG neurones were also analyzed in Spinal Cord Slice preparations. At clinical doses (5 mg/kg), propofol reversibly depressed action potentials elicited by noxious mechanical stimuli applied to the skin in the majority (6/8) of SG neurons reCorded under in vivo conditions. This depression may have been caused by interactions of propofol with GABA(A) receptors, as decay time of GABAergic sIPSCs was prolonged after propofol injection (128 +/- 11% of control, n = 5) with minimal effect on EPSCs. Although prolongation of IPSCs in vivo was reversible, IPSCs were progressively prolonged even after washout of propofol when the effect was tested using Spinal Cord Slices. Propofol had a mild depressant effect on Adelta- and C-afferent-mediated EPSCs. We conclude that systemic bolus injection of propofol reversibly depressed nociceptive transmission, at least in part, by enhancing postsynaptic GABA(A) receptor-mediated responses in the SG.

  • α2 adrenoceptor mediated presynaptic inhibition of primary afferent glutamatergic transmission in rat substantia gelatinosa neurons
    Anesthesiology, 2003
    Co-Authors: Yasuhiko Kawasaki, Hidemasa Furue, Eiichi Kumamoto, Megumu Yoshimura
    Abstract:

    Background: Although intrathecal administration of norepinephrine is known to produce analgesia, cellular mechanisms for this action have not yet been fully understood. Methods: The actions of norepinephrine (50 microm) on glutamatergic transmission were examined by using the whole cell patch clamp technique in substantia gelatinosa neurons of an adult rat Spinal Cord Slice with an attached dorsal root. Results: Norepinephrine inhibited the amplitude of monosynaptically evoked A delta-fiber and C-fiber excitatory postsynaptic currents in a reversible manner. When compared in magnitude between the A delta-fiber and C-fiber excitatory postsynaptic currents, the former inhibition (50 +/- 4%, n = 20) was significantly larger than the latter one (28 +/- 4%, n = 8). Both actions of norepinephrine were mimicked by an alpha2 adrenoceptor agonist, clonidine (10 microm), and an alpha 2A agonist, oxymetazoline (10 microm), but not by an alpha1 agonist, phenylephrine (10 microm), and a beta agonist, isoproterenol (40 microm). The inhibitory actions were antagonized by an alpha 2 antagonist, yohimbine (1 microm), all of the results of which indicate an involvement of alpha 2 adrenoceptors. Norepinephrine did not affect the amplitude of miniature excitatory postsynaptic current and of a response of substantia gelatinosa neurons to AMPA, indicating that its action on evoked excitatory postsynaptic currents is presynaptic in origin. Conclusions: Norepinephrine inhibits A delta-fiber- and C-fiber-mediated sensory transmission to substantia gelatinosa neurons through the activation of the alpha 2 adrenoceptor (possibly alpha2A type, based on the current, published behavioral and anatomical data) existing in primary afferent terminals; this action of norepinephrine is more effective in A delta-fiber than C-fiber transmission. This could contribute to at least a part of inhibitory modulation of pain sensation in the substantia gelatinosa by intrathecally administered norepinephrine.

  • voltage clamp reCordings of postsynaptic currents in substantia gelatinosa neurons in vitro and its applications to assess synaptic transmission
    Brain Research Protocols, 2001
    Co-Authors: Kun Yang, Hidemasa Furue, Megumu Yoshimura, Eiichi Kumamoto
    Abstract:

    We describe here procedures for reCording postsynaptic currents in substantia gelatinosa neurons on a transverse Spinal Cord Slice preparation with an attached dorsal root. At the holding potential of -70 mV, glutamatergic spontaneous excitatory postsynaptic currents (EPSCs) and dorsal root (A delta and/or C fiber) stimulation-evoked EPSCs could be observed. Whereas at the holding potential of 0 mV, spontaneous inhibitory postsynaptic currents (IPSCs) and dorsal root A delta fiber stimulation-evoked IPSCs could be encountered. The methods make it possible to evaluate synaptic transmission by analysing the postsynaptic currents on dorsal root attached Spinal Cord Slice.

Nils P Hailer - One of the best experts on this subject based on the ideXlab platform.

  • ventral horn neurons in mouse organotypic Spinal Cord Slice cultures are sustained to initial levels after eight days in vitro when treated with a combination of nt3 and il1ra
    Orthopaedic Proceedings, 2018
    Co-Authors: Nikos Schizas, Brittmarie Andersson, Nils P Hailer
    Abstract:

    Summary StatementSpinal Cord injury is characterised by an inflammatory cascade that leads to neuronal death by neurotoxicity. In a model of Spinal Cord damage we successfully preserved the number of ventral horn neurons by treatment with interleukin-1 receptor antagonist (IL1RA) and neurotrophin (NT)-3.IntroductionSecondary damage after Spinal Cord injury (SCI) is characterised by activation of microglial cells that release neurotoxic agents. This results in apoptotic death of neurons that survived the initial trauma. Interleukin (IL)-1 is one of the most prominent mediators of neurotoxicity. Organotypic Spinal Cord Slice cultures (OSCSC) are a useful in vitro model of Spinal Cord injury. We have previously shown that OSCSC degenerate substantially during in vitro incubation under standard conditions. Our aim was to treat OSCSC with the putatively neuroprotective agents IL-1 receptor antagonist (IL1RA) and neurotrophin (NT)-3 and to evaluate neuronal and microglial populations as well as axonal preservat...

  • neural crest stem cells protect Spinal Cord Slice cultures from excitotoxic neuronal damage and inhibit glial activation
    2015
    Co-Authors: Nikos Schizas, Brittmarie Andersson, Niclas Konig, Svitlana Vasylovska, Jan Hoeber, Elena N Kozlova, Nils P Hailer
    Abstract:

    The overall aim of this thesis was to establish strategies to minimize secondary damage to the injured Spinal Cord. Secondary damage that follows Spinal Cord injury (SCI) involves inflammatory and excitotoxic pathways. Regulation of these pathways using immunomodulatory and neuroprotective substances potentially protects the injured Spinal Cord from further damage. We also developed and studied resorbable biomaterials to be used as carriers for potential neuroprotectants to the injured Spinal Cord.We used transversal Spinal Cord Slice cultures (SCSCs) derived from postnatal mice as a model. SCSCs were maintained on different biomaterials and were studied after treatment with immunomodulatory and/or neurotrophic factors. They were further excitotoxically injured and subsequently treated with interleukin-1 receptor antagonist (IL1RA) or by neural crest stem cell (NCSC)-transplantation.The results show that biocompatible and resorbable hydrogels based on hyaluronic acid (HA) preserved neurons in SCSCs to a much higher extent than a conventional collagen-based biomaterial or standard polyethylene terephthalate (PET) membrane inserts. Glial activation was limited in the cultures maintained on HA-based hydrogel. The anti-inflammatory factor IL1RA protected SCSCs from degenerative mechanisms that occur during in vitro incubation, and IL1RA also protected SCSCs from excitotoxic injury induced by N-Methyl-d-Aspartate (NMDA). IL1RA specifically protected neurons that resided in the ventral horn, while other neuronal populations such as dorsal horn neurons and Renshaw cells did not respond to treatment. Finally, transplantation of NCSCs onto excitotoxically injured SCSCs protected from neuronal loss, apoptosis and glial activation, while NCSCs remained undifferentiated.The results presented in this thesis indicate that carriers based on HA seem to be more suitable than conventional collagen-based biomaterials since they enhance neuronal survival per se. The observed neuroprotection is likely due to biomechanical properties of HA. IL1RA protects SCSCs from spontaneous degeneration and from NMDA-induced injury, suggesting that excitotoxic mechanisms can be modulated through anti-inflammatory pathways. Different neuronal populations are affected by IL1RA to various degrees, suggesting that a combination of different neuroprotectants should be used in treatment strategies after SCI. Finally, NCSCs seem to protect SCSCs from excitotoxic injury through paracrine actions, since they remain undifferentiated and do not migrate into the tissue during in vitro incubation.It seems that combinations of neuroprotectants and carrier substances should be considered rather than one single strategy when designing future treatments for SCI. Incorporation of neuroprotectants such as IL1RA combined with stem cells in injectable biocompatible carriers based on HA is the final goal of our group in the treatment of SCI.

  • interleukin 1 receptor antagonist promotes survival of ventral horn neurons and suppresses microglial activation in mouse Spinal Cord Slice cultures
    Journal of Neuroscience Research, 2014
    Co-Authors: Nikos Schizas, Brittmarie Andersson, Jons Hilborn, Nils P Hailer
    Abstract:

    Secondary damage after Spinal Cord injury (SCI) induces neuronal demise through neurotoxicity and inflammation, and interleukin (IL)-1b is a key inflammatory mediator. We hypothesized that IL-1b is released in Spinal Cord Slice cultures (SCSC) and aimed at preventing the potentially neurotoxic effects of IL-1b by using interleukin-1 receptor antagonist (IL1RA). We hypothesized that IL1RA treatment enhances neuronal survival and suppresses microglial activation. SCSC were cultured up to 8 days in vitro (DIV) in the presence of IL1RA or without, either combined with trophic support using neurotrophin (NT)-3 or not. Four groups were studied: negative control, IL1RA, NT-3, and IL1RA 1NT-3. IL-1b concentrations in supernatants were measured by ELISA. SCSC were immunohistochemically stained for NeuN and a-neurofilament, and microglial cells were visualized with isolectin B4. After 8 DIV, ventral horn neurons were significantly more numerous in the IL1RA, NT-3, and IL1RA 1NT-3 groups compared with negative controls. Activated microglial cells were significantly less numerous in the IL1RA, NT-3, and IL1RA 1NT-3 groups compared with negative controls. Axons expanded into the collagen matrix after treatment with IL1RA, NT-3, or IL1RA 1NT-3, but not in negative controls. IL-1b release from cultures peaked after 6 hr and was lowest in the IL1RA 1NT-3 group. We conclude that IL-1b is released in traumatized Spinal Cord tissue and that IL1RA could exert its neuroprotective actions by blocking IL-1receptors. IL1RA thereby sustains neuronal survival irrespective of the presence of additional trophic support. Microglial activation is suppressed in the presence of IL1RA, suggesting decreased inflammatory activity. IL1RA treatment approaches may have substantial impact fol

  • ventral horn neurons in mouse organotypic Spinal Cord Slice cultures are sustained to initial levels after eight days in vitro when treated with a combination of nt3 and il1ra
    Journal of Bone and Joint Surgery-british Volume, 2014
    Co-Authors: Nikos Schizas, Brittmarie Andersson, Nils P Hailer
    Abstract:

    Summary Statement Spinal Cord injury is characterised by an inflammatory cascade that leads to neuronal death by neurotoxicity. In a model of Spinal Cord damage we successfully preserved the number of ventral horn neurons by treatment with interleukin-1 receptor antagonist (IL1RA) and neurotrophin (NT)-3. Introduction Secondary damage after Spinal Cord injury (SCI) is characterised by activation of microglial cells that release neurotoxic agents. This results in apoptotic death of neurons that survived the initial trauma. Interleukin (IL)-1 is one of the most prominent mediators of neurotoxicity. Organotypic Spinal Cord Slice cultures (OSCSC) are a useful in vitro model of Spinal Cord injury. We have previously shown that OSCSC degenerate substantially during in vitro incubation under standard conditions. Our aim was to treat OSCSC with the putatively neuroprotective agents IL-1 receptor antagonist (IL1RA) and neurotrophin (NT)-3 and to evaluate neuronal and microglial populations as well as axonal preservation. We hypothesised that treatment with the above substances would enhance neuronal survival and suppress microglial activation. Materials & Methods OSCSC were obtained from p9 (p=postnatal) mice and cultured in a 3-D collagen matrix for 0, 2, 4, 6 and 8 days in vitro (div). Neuroprotective substances were added to culture media, resulting in 4 treatment groups: IL1RA, NT3, IL1RA+NT3, and control (only medium). After fixation cultures were stained immunohistochemically for the neuronal marker NeuN and α-neurofilament (NF-L). Microglial cells were marked with isolectin B 4 (IB 4 ). Neurons in the ventral and dorsal horns were counted manually. The number of resting and activated microglial cells was calculated within the white and grey matter based on staining intensity and circularity index. Axonal preservation was evaluated qualitatively. Results OSCSC under control conditions showed signs of early degeneration after 2 div with decreased number of neurons within both the ventral and dorsal horns. However, significant differences between the groups were noted after 8 div: In the ventral horns, the neurons in all treatment groups were significantly more numerous compared to the control group. In the IL1RA+NT3 group the number of neurons did not differ significantly when compared to directly fixed cultures (div 0), whereas that number was significantly decreased in the other groups. After 8 div the number of activated microglial cells was significantly increased in the control group compared to all treatment groups both in the white and grey matter. Qualitative analysis of axonal preservation after 6 and 8 div revealed axonal sprouting within the white matter in all groups. However, these sprouting axons seemed to expand into the collagen matrix only in the three treatment groups. Discussion/Conclusion We demonstrate a neuroprotective effect of IL1RA and NT3 in OSCSC. OSCSC normally degenerate after in vitro incubation, however neurons in the ventral horns are sustained at initial levels in the IL1RA+NT3 group. At the same time microglial activation is suppressed in all treatment groups compared to controls. Finally, treatment of OSCSC with IL1RA and NT3 seem to be associated with axonal sprouting outside the cultures.

  • hyaluronic acid based hydrogel enhances neuronal survival in Spinal Cord Slice cultures from postnatal mice
    Journal of Biomaterials Applications, 2014
    Co-Authors: Nikos Schizas, Ramiro Rojas, Sujit Kootala, Brittmarie Andersson, Jennie Pettersson, Jons Hilborn, Nils P Hailer
    Abstract:

    Numerous biomaterials based on extracellular matrix-components have been developed. It was our aim to investigate whether a hyaluronic acid–based hydrogel improves neuronal survival and tissue preservation in organotypic Spinal Cord Slice cultures. Organotypic Spinal Cord Slice cultures were cultured for 4 days in vitro (div), either on hyaluronic acid–based hydrogel (hyaluronic acid–gel group), collagen gel (collagen group), directly on polyethylene terephthalate membrane inserts (control group), or in the presence of soluble hyaluronic acid (soluble hyaluronic acid group). Cultures were immunohistochemically stained against neuronal antigen NeuN and analyzed by confocal laser scanning microscopy. Histochemistry for choline acetyltransferance, glial fibrillary acidic protein, and Griffonia simplicifolia isolectin B4 followed by quantitative analysis was performed to assess motorneurons and different glial populations. Confocal microscopic analysis showed a 4-fold increase in the number of NeuN-positive n...

Kenneth L Tyler - One of the best experts on this subject based on the ideXlab platform.

  • minocycline has anti inflammatory effects and reduces cytotoxicity in an ex vivo Spinal Cord Slice culture model of west nile virus infection
    Journal of Virology, 2017
    Co-Authors: Eamon D Quick, Penny Clarke, Scott Seitz, Kenneth L Tyler
    Abstract:

    West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after WNV infection, leading to entry of activated peripheral immune cells. We utilized ex vivo Spinal Cord Slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the upregulation of proinflammatory genes and proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including interleukin 4 (IL-4), IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected Slices and markedly decreased levels of inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in ex vivo CNS tissue.IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available. Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Proinflammatory responses during WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following WNV infection have not been investigated. Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC while inhibiting the expression of genes associated with proinflammatory (M1) macrophage activation and was protective for multiple CNS cell types, indicating its potential use as a therapeutic reagent. This ex vivo culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, and microglia) to respond to minocycline and to modulate the inflammatory environment and cytotoxicity in response to WNV infection without peripheral immune cell involvement.

  • activation of intrinsic immune responses and microglial phagocytosis in an ex vivo Spinal Cord Slice culture model of west nile virus infection
    Journal of Virology, 2014
    Co-Authors: Eamon D Quick, J S Leser, Penny Clarke, Kenneth L Tyler
    Abstract:

    West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or Spinal Cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those that are dependent on infiltrating inflammatory cells has proven difficult. We used a murine ex vivo Spinal Cord Slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days after ex vivo infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV, resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection, as exemplified by their amoeboid morphology, the development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of proinflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and interleukin-6 (IL-6). The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced proinflammatory cytokine/chemokine expression profile, with inhibited production of CCL5, CCL2, and IL-6. Our findings establish that CNS-resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses. IMPORTANCE There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a Spinal Cord Slice culture (SCSC) model facilitates the study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses, including microglial activation and proinflammatory cytokine/chemokine production, develop independently of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.

Eiichi Kumamoto - One of the best experts on this subject based on the ideXlab platform.

  • α2 adrenoceptor mediated presynaptic inhibition of primary afferent glutamatergic transmission in rat substantia gelatinosa neurons
    Anesthesiology, 2003
    Co-Authors: Yasuhiko Kawasaki, Hidemasa Furue, Eiichi Kumamoto, Megumu Yoshimura
    Abstract:

    Background: Although intrathecal administration of norepinephrine is known to produce analgesia, cellular mechanisms for this action have not yet been fully understood. Methods: The actions of norepinephrine (50 microm) on glutamatergic transmission were examined by using the whole cell patch clamp technique in substantia gelatinosa neurons of an adult rat Spinal Cord Slice with an attached dorsal root. Results: Norepinephrine inhibited the amplitude of monosynaptically evoked A delta-fiber and C-fiber excitatory postsynaptic currents in a reversible manner. When compared in magnitude between the A delta-fiber and C-fiber excitatory postsynaptic currents, the former inhibition (50 +/- 4%, n = 20) was significantly larger than the latter one (28 +/- 4%, n = 8). Both actions of norepinephrine were mimicked by an alpha2 adrenoceptor agonist, clonidine (10 microm), and an alpha 2A agonist, oxymetazoline (10 microm), but not by an alpha1 agonist, phenylephrine (10 microm), and a beta agonist, isoproterenol (40 microm). The inhibitory actions were antagonized by an alpha 2 antagonist, yohimbine (1 microm), all of the results of which indicate an involvement of alpha 2 adrenoceptors. Norepinephrine did not affect the amplitude of miniature excitatory postsynaptic current and of a response of substantia gelatinosa neurons to AMPA, indicating that its action on evoked excitatory postsynaptic currents is presynaptic in origin. Conclusions: Norepinephrine inhibits A delta-fiber- and C-fiber-mediated sensory transmission to substantia gelatinosa neurons through the activation of the alpha 2 adrenoceptor (possibly alpha2A type, based on the current, published behavioral and anatomical data) existing in primary afferent terminals; this action of norepinephrine is more effective in A delta-fiber than C-fiber transmission. This could contribute to at least a part of inhibitory modulation of pain sensation in the substantia gelatinosa by intrathecally administered norepinephrine.

  • voltage clamp reCordings of postsynaptic currents in substantia gelatinosa neurons in vitro and its applications to assess synaptic transmission
    Brain Research Protocols, 2001
    Co-Authors: Kun Yang, Hidemasa Furue, Megumu Yoshimura, Eiichi Kumamoto
    Abstract:

    We describe here procedures for reCording postsynaptic currents in substantia gelatinosa neurons on a transverse Spinal Cord Slice preparation with an attached dorsal root. At the holding potential of -70 mV, glutamatergic spontaneous excitatory postsynaptic currents (EPSCs) and dorsal root (A delta and/or C fiber) stimulation-evoked EPSCs could be observed. Whereas at the holding potential of 0 mV, spontaneous inhibitory postsynaptic currents (IPSCs) and dorsal root A delta fiber stimulation-evoked IPSCs could be encountered. The methods make it possible to evaluate synaptic transmission by analysing the postsynaptic currents on dorsal root attached Spinal Cord Slice.

  • alteration in synaptic inputs through c afferent fibers to substantia gelatinosa neurons of the rat Spinal dorsal horn during postnatal development
    Neuroscience, 2000
    Co-Authors: Terumasa Nakatsuka, Eiichi Kumamoto, Toyofumi Ataka, Tetsuya Tamaki, Megumu Yoshimura
    Abstract:

    Abstract The change in synaptic inputs through primary afferent C- and A-fibers during postnatal development was examined in substantia gelatinosa neurons of a rat Spinal Cord Slice with an attached L5 dorsal root by use of the blind whole-cell patch-clamp technique; the synaptic responses were compared between the Slices obtained from immature (postnatal days 21–23) and mature (postnatal days 56–60) male rats. The mono- and/or polysynaptic afferent inputs were monitored by reCording glutamatergic excitatory postsynaptic currents and potentials evoked by stimulating C- and A-fibers, the identification of which was based on the values of threshold stimulus intensity and of the conduction velocity of the fibers, determined by intracellular reCordings from dorsal root ganglion neurons. Immature substantia gelatinosa neurons received synaptic inputs through Aβ-, Aδ- and C-afferents, with proportions of 51%, 46% and 36%, respectively. In mature substantia gelatinosa neurons, C- and Aδ-afferent inputs were increased in number (to 84% and 86%, respectively), while Aβ-inputs were decreased to 9%. In both immature and mature rats, repetitive stimulation of C-afferents did not elicit any slow responses, which are longer in duration than the monosynaptic excitatory postsynaptic currents, although C-fibers are known to contain not only excitatory amino acids, but also neuropeptides such as substance P, which is thought to be involved in the production of slow responses. These results indicate that both C- and Aδ-afferents innervating substantia gelatinosa neurons are reorganized following maturation, accompanied by a withdrawal or elimination of Aβ-fibers from the substantia gelatinosa, probably due to a competition among the fibers during development. In spite of the developmental increase in C-fiber inputs, mature as well as immature substantia gelatinosa neurons did not display any slow synaptic responses, which appear to be mediated by transmitters other than excitatory amino acids.

  • baclofen inhibits more effectively c afferent than aδ afferent glutamatergic transmission in substantia gelatinosa neurons of adult rat Spinal Cord Slices
    Pain, 2000
    Co-Authors: Toyofumi Ataka, Eiichi Kumamoto, Koki Shimoji, Megumu Yoshimura
    Abstract:

    Although intrathecal administration of baclofen, a selective GABAB-receptor agonist, is known to have an antinociceptive effect on various pain models, the role of presynaptic GABAB receptors in antinociception is not well characterized. In the present study, the action of baclofen on primary afferent-evoked glutamatergic excitatory transmission was examined in substantia gelatinosa (SG) neurons of an adult rat Spinal Cord Slice with an attached dorsal root, prepared from the lumbar segment, by use of the blind whole-cell patch-clamp technique. Under the condition where a postsynaptic action of baclofen was inhibited, baclofen (1 μM) reduced the amplitudes of excitatory postsynaptic currents (EPSCs; VH=−70 mV) which were monosynaptically evoked by stimulating primary-afferent C- and/or Aδ-fibers and which were remarkably depressed by CNQX (10 μM). The identification of the C-fiber or Aδ-fiber EPSC was based on antidromic action potentials reCorded from neurons of isolated dorsal root ganglia. The C-fiber EPSC was depressed in peak amplitude by baclofen (1 μM) to a larger extent than the Aδ-fiber EPSC (20 and 45% of control, respectively). Each of the baclofen actions was suppressed by a selective GABAB-receptor antagonist, CGP 35348 (50 μM). Baclofen (1 μM) did not affect a response of SG neurons to bath-applied AMPA (10 μM). These results indicate that baclofen inhibits the release of L-glutamate from Aδ and C primary-afferent terminals in the SG through the activation of GABAB receptor; this action is more effective to C-fiber than Aδ-fiber transmission. Considering that the SG is the main part of termination of Aδ- and C-fibers transmitting nociceptive information, the present finding would account for at least a part of the inhibitory action of baclofen on pain transmission.

Eamon D Quick - One of the best experts on this subject based on the ideXlab platform.

  • minocycline has anti inflammatory effects and reduces cytotoxicity in an ex vivo Spinal Cord Slice culture model of west nile virus infection
    Journal of Virology, 2017
    Co-Authors: Eamon D Quick, Penny Clarke, Scott Seitz, Kenneth L Tyler
    Abstract:

    West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after WNV infection, leading to entry of activated peripheral immune cells. We utilized ex vivo Spinal Cord Slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the upregulation of proinflammatory genes and proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including interleukin 4 (IL-4), IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected Slices and markedly decreased levels of inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in ex vivo CNS tissue.IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available. Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Proinflammatory responses during WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following WNV infection have not been investigated. Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC while inhibiting the expression of genes associated with proinflammatory (M1) macrophage activation and was protective for multiple CNS cell types, indicating its potential use as a therapeutic reagent. This ex vivo culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, and microglia) to respond to minocycline and to modulate the inflammatory environment and cytotoxicity in response to WNV infection without peripheral immune cell involvement.

  • activation of intrinsic immune responses and microglial phagocytosis in an ex vivo Spinal Cord Slice culture model of west nile virus infection
    Journal of Virology, 2014
    Co-Authors: Eamon D Quick, J S Leser, Penny Clarke, Kenneth L Tyler
    Abstract:

    West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or Spinal Cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those that are dependent on infiltrating inflammatory cells has proven difficult. We used a murine ex vivo Spinal Cord Slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days after ex vivo infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV, resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection, as exemplified by their amoeboid morphology, the development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of proinflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and interleukin-6 (IL-6). The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced proinflammatory cytokine/chemokine expression profile, with inhibited production of CCL5, CCL2, and IL-6. Our findings establish that CNS-resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses. IMPORTANCE There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a Spinal Cord Slice culture (SCSC) model facilitates the study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses, including microglial activation and proinflammatory cytokine/chemokine production, develop independently of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.