A novel primary culture method for high-purity satellite glial cells derived from rat dorsal root ganglion
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摘要
Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture method for satellite glial cells derived from rat dorsal root ganglia requires no digestion skill. Hence, the aim of the present study was to establish a novel primary culture method for satellite glial cells derived from dorsal root ganglia. Neonatal rat spine was collected and an incision made to expose the transverse protrusion and remove dorsal root ganglia. Dorsal root ganglia were freed from nerve fibers, connective tissue, and capsule membranes, then rinsed and transferred to 6-well plates, and cultured in a humidified 5% CO_2 incubator at 37°C. After 3 days in culture, some cells had migrated from dorsal root ganglia. After subculture, cells were identified by immunofluorescence labeling for three satellite glial cell-specific markers: glutamine synthetase, glial fibrillary acidic protein, and S100β. Cultured cells expressed glutamine synthetase, glial fibrillary acidic protein, and S100β, suggesting they are satellite glial cells with a purity of > 95%. Thus, we have successfully established a novel primary culture method for obtaining high-purity satellite glial cells from rat dorsal root ganglia without digestion.
Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture method for satellite glial cells derived from rat dorsal root ganglia requires no digestion skill. Hence, the aim of the present study was to establish a novel primary culture method for satellite glial cells derived from dorsal root ganglia. Neonatal rat spine was collected and an incision made to expose the transverse protrusion and remove dorsal root ganglia. Dorsal root ganglia were freed from nerve fibers, connective tissue, and capsule membranes, then rinsed and transferred to 6-well plates, and cultured in a humidified 5% CO_2 incubator at 37°C. After 3 days in culture, some cells had migrated from dorsal root ganglia. After subculture, cells were identified by immunofluorescence labeling for three satellite glial cell-specific markers: glutamine synthetase, glial fibrillary acidic protein, and S100β. Cultured cells expressed glutamine synthetase, glial fibrillary acidic protein, and S100β, suggesting they are satellite glial cells with a purity of > 95%. Thus, we have successfully established a novel primary culture method for obtaining high-purity satellite glial cells from rat dorsal root ganglia without digestion.
Satellite glial cells surround neurons within dorsal root ganglia. Previous studies have focused on single-cell suspensions of cultured neurons derived from rat dorsal root ganglia. At present, the primary culture method for satellite glial cells derived from rat dorsal root ganglia requires no digestion skill. Hence, the aim of the present study was to establish a novel primary culture method for satellite glial cells derived from dorsal root ganglia. Neonatal rat spine was collected and an incision made to expose the transverse protrusion and remove dorsal root ganglia. Dorsal root ganglia were freed from nerve fibers, connective tissue, and capsule membranes, then rinsed and transferred to 6-well plates, and cultured in a humidified 5% CO_2 incubator at 37°C. After 3 days in culture, some cells had migrated from dorsal root ganglia. After subculture, cells were identified by immunofluorescence labeling for three satellite glial cell-specific markers: glutamine synthetase, glial fibrillary acidic protein, and S100β. Cultured cells expressed glutamine synthetase, glial fibrillary acidic protein, and S100β, suggesting they are satellite glial cells with a purity of > 95%. Thus, we have successfully established a novel primary culture method for obtaining high-purity satellite glial cells from rat dorsal root ganglia without digestion.
引文
Bai L,Wang X,Li Z,Kong C,Zhao Y,Qian JL,Kan Q,Zhang W,Xu JT(2016)Upregulation of chemokine CXCL12 in the dorsal root ganglia and spinal cord contributes to the development and maintenance of neuropathic pain following spared nerve injury in rats.Neurosci Bull 32:27-40.
Bierlein De la Rosa M,Sharma AD,Mallapragada SK,Sakaguchi DS(2017)Transdifferentiation of brain-derived neurotrophic factor(BDNF)-secreting mesenchymal stem cells significantly enhance BDNF secretion and Schwann cell marker proteins.J Biosci Bioeng124:572-582.
Capuano A,De Corato A,Lisi L,Tringali G,Navarra P,Dello Russo C(2009)Proinflammatory-activated trigeminal satellite cells promote neuronal sensitization:relevance for migraine pathology.Mol Pain5:43.
Duobles T,Lima Tde S,Levy Bde F,Chadi G(2008)S100beta and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury.Acta Cir Bras 23:555-560.
Fang L,Wang YN,Cui XL,Fang SY,Ge JY,Sun Y,Liu ZH(2012)The role and mechanism of action of activin A in neurite outgrowth of chicken embryonic dorsal root ganglia.J Cell Sci 125:1500-1507.
Fei W,Xiang H,Fischer G,Zhen L,Dupont MJ,Hogan QH,Yu H(2016)HMG-CoA synthase isoenzymes 1 and 2 localize to satellite glial cells in dorsal root ganglia and are differentially regulated by peripheral nerve injury.Brain Res 1652:62-70.
Feldman-Goriachnik R,Wu B,Hanani M(2018)Cholinergic responses of satellite glial cells in the superior cervical ganglia.Neurosci Lett 671:19-24.
Fornaro M,Sharthiya H,Tiwari V(2018)Adult mouse DRG explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection.J Vis Exp doi:10.3791/56757.
Fregoso SP,Hoover DB(2012)Development of cardiac parasympathetic neurons,glial cells,and regional cholinergic innervation of the mouse heart.Neuroscience 221:28-36.
Fujiwara S,Hoshikawa S,Ueno T,Hirata M,Saito T,Ikeda T,Kawaguchi H,Nakamura K,Tanaka S,Ogata T(2014)SOX10 transactivates S100B to suppress Schwann cell proliferation and to promote myelination.PLoS One 9:e115400.
Goto T,Iwai H,Kuramoto E,Yamanaka A(2017)Neuropeptides and ATP signaling in the trigeminal ganglion.Jpn Dent Sci Rev 53:117-124.
Gu Y,Chen Y,Zhang X,Li GW,Wang C,Huang LYM(2010)Neuronal soma-satellite glial cell interactions in sensory ganglia and the participation of purinergic receptors.Neuron Glia Biol 6:53-62.
Hanani M(2010)Satellite glial cells in sympathetic and parasympathetic ganglia:in search of function.Brain Res Rev 64:304-327.
Hossain MZ,Unno S,Ando H,Masuda Y,Kitagawa J(2017)Neuron-glia crosstalk and neuropathic pain:involvement in the modulation of motor activity in the orofacial region.Int J Mol Sci.18.pii:E2051.
Komiya H,Shimizu K,Noma N,Tsuboi Y,Honda K,Kanno K,Ohara K,Shinoda M,Ogiso B,Iwata K(2018)Role of neuron-glial interaction mediated by IL-1βin ectopic tooth pain.J Dent Res 97:467-475.
Lagares A,Li HY,Zhou XF,Avenda?o C(2007)Primary sensory neuron addition in the adult rat trigeminal ganglion:evidence for neural crest glio-neuronal precursor maturation.J Neurosci 27:7939-7953.
Li HY,Zhou XF(2008)Potential conversion of adult clavicle-derived chondrocytes into neural lineage cells in vitro.J Cell Physiol214:630-644.
Li HY,Say EH,Zhou XF(2007)Isolation and characterization of neural crest progenitors from adult dorsal root ganglia.Stem Cells25:2053-2065.
Li J,Ouyang Q,Chen CW,Chen QB,Li XN,Xiang ZH,Yuan HB(2017)Neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleavage of E-cadherin in satellite glial cells.Pain Med 18:1752-1766.
Liu H,Zhao L,Gu W,Liu Q,Gao Z,Zhu X,Wu Z,He H,Huang F,Fan W(2018)Activation of satellite glial cells in trigeminal ganglion following dental injury and inflammation.J Mol Histol 49:257-263.
Owen DE,Egerton J(2012)Culture of dissociated sensory neurons from dorsal root ganglia of postnatal and adult rats.Methods Mol Biol 846:179-187.
Pannese E,Ledda M,Cherkas PS,Huang TY,Hanani M(2003)Satellite cell reactions to axon injury of sensory ganglion neurons:Increase in number of gap junctions and formation of bridges connecting previously separate perineuronal sheaths.Anat Embryol(Berl)206:337-347.
Pi?ero G,Usach V,Soto PA,Monje PV,Setton-Avruj P(2018)EGFPtransgene:a useful tool to track transplanted bone marrow mononuclear cell contribution to peripheral remyelination.Transgenic Res 27:135-153.
Retamal MA,Riquelme MA,Stehberg J,Alcayaga J(2017)Connexin43 hemichannels in satellite glial cells,can they influence sensory neuron activity?Front Mol Neurosci 10:374.
Spray DC,Hanani M(2017)Gap junctions,pannexins and pain.Neurosci Lett pii:S0304-3940(17)30515-3.
Takeda M,Takahashi M,Matsumoto S(2009)Contribution of the activation of satellite glia in sensory ganglia to pathological pain.Neurosci Biobehav Rev 33:784-792.
Tongtako W,Lehmbecker A,Wang Y,Hahn K,Baumg?rtner W,Gerhauser I(2017)Canine dorsal root ganglia satellite glial cells represent an exceptional cell population with astrocytic and oligodendrocytic properties.Sci Rep 7:13915.
Yamakita S,Horii Y,Takemura H,Matsuoka Y,Yamashita A,Yamaguchi Y,Matsuda M,Sawa T,Amaya F(2018)Synergistic activation of ERK1/2 between A-fiber neurons and glial cells in the DRGcontributes to pain hypersensitivity after tissue injury.Mol Pain14:1744806918767508.
Yi Z,Xie L,Zhou C,Yuan H,Ouyang S,Fang Z,Zhao S,Jia T,Zou L,Wang S,Xue Y,Wu B,Gao Y,Li G,Liu S,Xu H,Xu C,Zhang C,Liang S(2018)P2Y12 receptor upregulation in satellite glial cells is involved in neuropathic pain induced by HIV glycoprotein 120 and2’,3’-dideoxycytidine.Purinergic Signal 14:47-58.
Zemel BM,Muqeem T(2017)Calcineurin dysregulation underlies spinal cord injury-induced K(+)channel dysfunction in DRG.Neurons 37:8256-8272.
Zhang MH,Hu YM,Zhang H,Hu YP,Liu QZ,Wang HC,Li WY(2012)Brain-derived neurotrophic factor expression in the spinal dorsal horn and dorsal root ganglion in a rat model of sciatic nerve injury after intrathecal transplantation of neural stem cells.Zhongguo Zuzhi Gongcheng Yanjiu 16:1851-1855.
Zhu G,Chen Z,Dai B,Zheng C,Jiang H,Xu Y,Sheng X,Guo J,Dan Y,Liang S,Li G(2018)Chronic lead exposure enhances the sympathoexcitatory response associated with P2X4 receptor in rat stellate ganglia.Environ Toxicol 33:631-639.
Bierlein De la Rosa M,Sharma AD,Mallapragada SK,Sakaguchi DS(2017)Transdifferentiation of brain-derived neurotrophic factor(BDNF)-secreting mesenchymal stem cells significantly enhance BDNF secretion and Schwann cell marker proteins.J Biosci Bioeng124:572-582.
Capuano A,De Corato A,Lisi L,Tringali G,Navarra P,Dello Russo C(2009)Proinflammatory-activated trigeminal satellite cells promote neuronal sensitization:relevance for migraine pathology.Mol Pain5:43.
Duobles T,Lima Tde S,Levy Bde F,Chadi G(2008)S100beta and fibroblast growth factor-2 are present in cultured Schwann cells and may exert paracrine actions on the peripheral nerve injury.Acta Cir Bras 23:555-560.
Fang L,Wang YN,Cui XL,Fang SY,Ge JY,Sun Y,Liu ZH(2012)The role and mechanism of action of activin A in neurite outgrowth of chicken embryonic dorsal root ganglia.J Cell Sci 125:1500-1507.
Fei W,Xiang H,Fischer G,Zhen L,Dupont MJ,Hogan QH,Yu H(2016)HMG-CoA synthase isoenzymes 1 and 2 localize to satellite glial cells in dorsal root ganglia and are differentially regulated by peripheral nerve injury.Brain Res 1652:62-70.
Feldman-Goriachnik R,Wu B,Hanani M(2018)Cholinergic responses of satellite glial cells in the superior cervical ganglia.Neurosci Lett 671:19-24.
Fornaro M,Sharthiya H,Tiwari V(2018)Adult mouse DRG explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection.J Vis Exp doi:10.3791/56757.
Fregoso SP,Hoover DB(2012)Development of cardiac parasympathetic neurons,glial cells,and regional cholinergic innervation of the mouse heart.Neuroscience 221:28-36.
Fujiwara S,Hoshikawa S,Ueno T,Hirata M,Saito T,Ikeda T,Kawaguchi H,Nakamura K,Tanaka S,Ogata T(2014)SOX10 transactivates S100B to suppress Schwann cell proliferation and to promote myelination.PLoS One 9:e115400.
Goto T,Iwai H,Kuramoto E,Yamanaka A(2017)Neuropeptides and ATP signaling in the trigeminal ganglion.Jpn Dent Sci Rev 53:117-124.
Gu Y,Chen Y,Zhang X,Li GW,Wang C,Huang LYM(2010)Neuronal soma-satellite glial cell interactions in sensory ganglia and the participation of purinergic receptors.Neuron Glia Biol 6:53-62.
Hanani M(2010)Satellite glial cells in sympathetic and parasympathetic ganglia:in search of function.Brain Res Rev 64:304-327.
Hossain MZ,Unno S,Ando H,Masuda Y,Kitagawa J(2017)Neuron-glia crosstalk and neuropathic pain:involvement in the modulation of motor activity in the orofacial region.Int J Mol Sci.18.pii:E2051.
Komiya H,Shimizu K,Noma N,Tsuboi Y,Honda K,Kanno K,Ohara K,Shinoda M,Ogiso B,Iwata K(2018)Role of neuron-glial interaction mediated by IL-1βin ectopic tooth pain.J Dent Res 97:467-475.
Lagares A,Li HY,Zhou XF,Avenda?o C(2007)Primary sensory neuron addition in the adult rat trigeminal ganglion:evidence for neural crest glio-neuronal precursor maturation.J Neurosci 27:7939-7953.
Li HY,Zhou XF(2008)Potential conversion of adult clavicle-derived chondrocytes into neural lineage cells in vitro.J Cell Physiol214:630-644.
Li HY,Say EH,Zhou XF(2007)Isolation and characterization of neural crest progenitors from adult dorsal root ganglia.Stem Cells25:2053-2065.
Li J,Ouyang Q,Chen CW,Chen QB,Li XN,Xiang ZH,Yuan HB(2017)Neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleavage of E-cadherin in satellite glial cells.Pain Med 18:1752-1766.
Liu H,Zhao L,Gu W,Liu Q,Gao Z,Zhu X,Wu Z,He H,Huang F,Fan W(2018)Activation of satellite glial cells in trigeminal ganglion following dental injury and inflammation.J Mol Histol 49:257-263.
Owen DE,Egerton J(2012)Culture of dissociated sensory neurons from dorsal root ganglia of postnatal and adult rats.Methods Mol Biol 846:179-187.
Pannese E,Ledda M,Cherkas PS,Huang TY,Hanani M(2003)Satellite cell reactions to axon injury of sensory ganglion neurons:Increase in number of gap junctions and formation of bridges connecting previously separate perineuronal sheaths.Anat Embryol(Berl)206:337-347.
Pi?ero G,Usach V,Soto PA,Monje PV,Setton-Avruj P(2018)EGFPtransgene:a useful tool to track transplanted bone marrow mononuclear cell contribution to peripheral remyelination.Transgenic Res 27:135-153.
Retamal MA,Riquelme MA,Stehberg J,Alcayaga J(2017)Connexin43 hemichannels in satellite glial cells,can they influence sensory neuron activity?Front Mol Neurosci 10:374.
Spray DC,Hanani M(2017)Gap junctions,pannexins and pain.Neurosci Lett pii:S0304-3940(17)30515-3.
Takeda M,Takahashi M,Matsumoto S(2009)Contribution of the activation of satellite glia in sensory ganglia to pathological pain.Neurosci Biobehav Rev 33:784-792.
Tongtako W,Lehmbecker A,Wang Y,Hahn K,Baumg?rtner W,Gerhauser I(2017)Canine dorsal root ganglia satellite glial cells represent an exceptional cell population with astrocytic and oligodendrocytic properties.Sci Rep 7:13915.
Yamakita S,Horii Y,Takemura H,Matsuoka Y,Yamashita A,Yamaguchi Y,Matsuda M,Sawa T,Amaya F(2018)Synergistic activation of ERK1/2 between A-fiber neurons and glial cells in the DRGcontributes to pain hypersensitivity after tissue injury.Mol Pain14:1744806918767508.
Yi Z,Xie L,Zhou C,Yuan H,Ouyang S,Fang Z,Zhao S,Jia T,Zou L,Wang S,Xue Y,Wu B,Gao Y,Li G,Liu S,Xu H,Xu C,Zhang C,Liang S(2018)P2Y12 receptor upregulation in satellite glial cells is involved in neuropathic pain induced by HIV glycoprotein 120 and2’,3’-dideoxycytidine.Purinergic Signal 14:47-58.
Zemel BM,Muqeem T(2017)Calcineurin dysregulation underlies spinal cord injury-induced K(+)channel dysfunction in DRG.Neurons 37:8256-8272.
Zhang MH,Hu YM,Zhang H,Hu YP,Liu QZ,Wang HC,Li WY(2012)Brain-derived neurotrophic factor expression in the spinal dorsal horn and dorsal root ganglion in a rat model of sciatic nerve injury after intrathecal transplantation of neural stem cells.Zhongguo Zuzhi Gongcheng Yanjiu 16:1851-1855.
Zhu G,Chen Z,Dai B,Zheng C,Jiang H,Xu Y,Sheng X,Guo J,Dan Y,Liang S,Li G(2018)Chronic lead exposure enhances the sympathoexcitatory response associated with P2X4 receptor in rat stellate ganglia.Environ Toxicol 33:631-639.