脊髓损伤模型大鼠软脊膜下注射2型腺相关病毒免疫荧光染色分析
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摘要
背景:经软脊膜下注射腺相关病毒的方法可将病毒局限于以注射点为中心的较小范围内,全身毒副作用小,节省病毒的用量,但目前尚无将此方法应用于脊髓损伤大鼠的相关报道,对损伤部位星形胶质细胞和神经细胞的转染情况尚不明确。目的:探讨通过软脊膜下注射2型腺相关病毒对脊髓损伤部位的星形胶质细胞和神经细胞的转染效果及手术过程的技术要点。方法:实验方案经吉林大学动物实验伦理委员会批准。24只Wistar大鼠随机分为2组:腺相关病毒组和模型组,均使用钳夹法构建脊髓损伤模型。腺相关病毒组经软脊膜下注射60μL病毒溶液;模型组经软脊膜下注射等量5%右旋糖酐溶液。于术后7,14和21d取脊髓标本,进行免疫荧光染色,观察星形胶质细胞分布情况和2型腺相关病毒转染情况。结果与结论:①经软脊膜下注射病毒后,病毒溶液最终均匀分布于损伤部位上下约1 cm的范围内;②术后7,14 d仅有微量绿色荧光蛋白在脊髓损伤区域表达,直至术后21 d,在脊髓损伤空洞周围可见有大量反应性星形胶质细胞,其在脊髓空洞周边部表达最为强烈,出现明显的胶质界膜;同时发现,2型腺相关病毒可稳定转染星形胶质细胞和神经细胞,其中星形胶质细胞集中分布于脊髓空洞周边部,而神经细胞散在分布于病毒转染范围内,注射范围以外正常组织仅见微量2型腺相关病毒转染;③该方法具有能将病毒局限于损伤部位,可靶向性转染星形胶质细胞和神经细胞,具有注射量病毒量低,全身毒副作用小,成本节约等优点。
BACKGROUND: The method of injecting adeno-associated virus subpial can limit the virus to a small area centered on the injection point,with less systemic toxicity and side effects, and save the amount of virus. However, there are no reports on the application of this method in spinal cord injury rats, and the transfection of astrocytes and neurons at the injured site is still unclear.OBJECTIVE: To investigate the transfection effect of subpial injection of adeno-associated virus 2 on astrocytes and neurons in the spinal cord injury site and the technical points of the surgical procedure.METHODS: The study was approved by the Experimental Animal Ethics Committee of Jilin University. Twenty-four Wistar rats were randomly divided into two groups: adeno-associated virus group and model group. Rats in both groups were used to establish the spinal cord injury model using the clamp method. The adeno-associated virus group received 60 μL of virus solution by subpial injection, and the model group received an equal amount of 5% dextran solution. At postoperative 7, 14, and 21 days, spinal cord specimens were taken for immunofluorescence staining to observe the distribution of astrocytes and the transfection of adeno-associated virus 2.RESULTS AND CONCLUSION:(1) After subpial injection of the virus, the virus solution evenly distributed in the range of about 1 cm above and below the injured site.(2) At 7 and 14 days after surgery, only a small amount of green fluorescent protein was expressed in the spinal cord injury area. Until day 21, a large number of reactive astrocytes were observed around the spinal cord injury cavity. The strongest expression was in the periphery of the spinal cord cavity, with obvious glial boundary membrane. It was also found that adeno-associated virus 2 could stably transfect astrocytes and neurons, in which astrocytes were concentrated around the periphery of the spinal cord cavity,while neurons were scattered within the scope of virus transfection. Only trace adeno-associated virus 2 transfection was found in normal tissues outside the scope of injection.(3) The method has the advantages of confining the virus to the injured site, targeting astrocyte and nerve cell transfection, low viral dosage, low systemic toxicity and side effects, and low cost.
BACKGROUND: The method of injecting adeno-associated virus subpial can limit the virus to a small area centered on the injection point,with less systemic toxicity and side effects, and save the amount of virus. However, there are no reports on the application of this method in spinal cord injury rats, and the transfection of astrocytes and neurons at the injured site is still unclear.OBJECTIVE: To investigate the transfection effect of subpial injection of adeno-associated virus 2 on astrocytes and neurons in the spinal cord injury site and the technical points of the surgical procedure.METHODS: The study was approved by the Experimental Animal Ethics Committee of Jilin University. Twenty-four Wistar rats were randomly divided into two groups: adeno-associated virus group and model group. Rats in both groups were used to establish the spinal cord injury model using the clamp method. The adeno-associated virus group received 60 μL of virus solution by subpial injection, and the model group received an equal amount of 5% dextran solution. At postoperative 7, 14, and 21 days, spinal cord specimens were taken for immunofluorescence staining to observe the distribution of astrocytes and the transfection of adeno-associated virus 2.RESULTS AND CONCLUSION:(1) After subpial injection of the virus, the virus solution evenly distributed in the range of about 1 cm above and below the injured site.(2) At 7 and 14 days after surgery, only a small amount of green fluorescent protein was expressed in the spinal cord injury area. Until day 21, a large number of reactive astrocytes were observed around the spinal cord injury cavity. The strongest expression was in the periphery of the spinal cord cavity, with obvious glial boundary membrane. It was also found that adeno-associated virus 2 could stably transfect astrocytes and neurons, in which astrocytes were concentrated around the periphery of the spinal cord cavity,while neurons were scattered within the scope of virus transfection. Only trace adeno-associated virus 2 transfection was found in normal tissues outside the scope of injection.(3) The method has the advantages of confining the virus to the injured site, targeting astrocyte and nerve cell transfection, low viral dosage, low systemic toxicity and side effects, and low cost.
引文
[1]Jendelova P.Therapeutic Strategies for Spinal Cord Injury.Int JMol Sci.2018;19(10).pii:E3200.
[2]Ahmed A,Patil AA,Agrawal DK.Immunobiology of spinal cord injuries and potential therapeutic approaches.Mol Cell Biochem.2018;441(1-2):181-189.
[3]Dalamagkas K,Tsintou M,Seifalian A,et al.Translational Regenerative Therapies for Chronic Spinal Cord Injury.Int JMol Sci.2018;19(6).pii:E1776.
[4]Ahuja CS,Nori S,Tetreault L,et al.Traumatic Spinal Cord Injury-Repair and Regeneration.Neurosurgery.2017;80(3):S9-S22.
[5]Huang X,Gu YK,Cheng XY,et al.[Astrocytes as therapeutic targets after spinal cord injury].Sheng Li Xue Bao.2017;69(6):794-804.
[6]李剑锋,闫金玉,夏润福,等.脊髓损伤胶质瘢痕形成及星形胶质细胞作用的研究与转化意义[J].中国组织工程研究,2016,20(37):5609-5616.
[7]Okada S,Hara M,Kobayakawa K,et al.Astrocyte reactivity and astrogliosis after spinal cord injury.Neurosci Res.2018;126:39-43.
[8]Sofroniew MV,Vinters HV.Astrocytes:biology and pathology.Acta Neuropathol.2010;119(1):7-35.
[9]Lu X,Xue P,Fu L,et al.HAX1 is associated with neuronal apoptosis and astrocyte proliferation after spinal cord injury.Tissue Cell.2018;54:1-9.
[10]Kameda T,Imamura T,Nakashima K.Epigenetic regulation of neural stem cell differentiation towards spinal cord regeneration.Cell Tissue Res.2018;371(1):189-199.
[11]Chen XQ,Chen C,Hao J,et al.Effect of CLIP3 Upregulation on Astrocyte Proliferation and Subsequent Glial Scar Formation in the Rat Spinal Cord via STAT3 Pathway After Injury.J Mol Neurosci.2018;64(1):117-128.
[12]Ramadan WS,Abdel-Hamid GA,Al-Karim S,et al.Neuroectodermal stem cells:A remyelinating potential in acute compressed spinal cord injury in rat model.J Biosci.2018;43(5):897-909.
[13]巩朝阳,刘开鑫,向高,等.脊髓损伤后胶质瘢痕形成的研究进展[J].中国康复理论与实践,2018,24(6):641-644.
[14]Song JL,Zheng W,Chen W,et al.Lentivirus-mediated microRNA-124 gene-modified bone marrow mesenchymal stem cell transplantation promotes the repair of spinal cord injury in rats.Exp Mol Med.201719;49(5):e332.
[15]Yin H,Shen LM,Xu C,et al.Lentivirus-Mediated Overexpression of mi R-29a Promotes Axonal Regeneration and Functional Recovery in Experimental Spinal Cord Injury via PI3K/Akt/mTORPathway.Neurochem Res.2018;43(11):2038-2046.
[16]Choudhury SR,Hudry E,Maguire CA,et al.Viral vectors for therapy of neurologic diseases.Neuropharmacology.2017;120:63-80.
[17]Saraiva J,Nobre RJ,De Almeida LP.Gene therapy for the CNSusing AAVs:The impact of systemic delivery by AAV9.JControl Release.2016;241:94-109.
[18]Gao GP,Vandenberghe LH,Alvira MR,et al.Clades of Adeno-associated viruses are widely disseminated in human tissues.J Virol.2004;78(12):6381-6388.
[19]陈如意,陈素峰,周丹,等.2型腺相关病毒作为基因治疗载体的研究进展[J].生命科学,2013,25(6):595-600.
[20]Davidson BL,Stein CS,Heth JA,et al.Recombinant adeno-associated virus type 2,4,and 5 vectors:transduction of variant cell types and regions in the mammalian central nervous system.Proc Natl Acad Sci U S A.2000;97(7):3428-3432.
[21]Murlidharan G,Samulski RJ,Asokan A.Biology of adenoassociated viral vectors in the central nervous system.Front Mol Neurosci.2014;7:76.
[22]Tadokoro T,Miyanohara A,Navarro M,et al.Subpial Adeno-associated Virus 9(AAV9)Vector Delivery in Adult Mice.J Vis Exp.2017;(125).doi:10.3791/55770.
[23]Gray SJ,Matagne V,Bachaboina L,et al.Preclinical Differences of Intravascular AAV9 Delivery to Neurons and Glia:AComparative Study of Adult Mice and Nonhuman Primates.Molecular Therapy.2011;19(6):1058-1069.
[24]Samaranch L,Salegio EA,San Sebastian W,et al.AdenoAssociated Virus Serotype 9 Transduction in the Central Nervous System of Nonhuman Primates.Human Gene Therapy.2012;23(4):382-389.
[25]Miyake N,Miyake K,Yamamoto M,et al.Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors.Brain Res.2011;1389:19-26.
[26]Povysheva T,Shmarov M,Logunov D,et al.Post-spinal cord injury astrocyte-mediated functional recovery in rats after intraspinal injection of the recombinant adenoviral vectors Ad5-VEGF and Ad5-ANG.J Neurosurg Spine.2017;27(1):105-115.
[27]李东卿.经肋间后动脉介入移植骨髓基质干细胞修复脊髓损伤的研究[D].广州:南方医科大学,2012.
[28]杞少华,王海峰,熊文平,等.骨髓间充质干细胞尾静脉注射与局部靶点注射对大鼠脊髓损伤修复效果的比较[J].中华实验外科杂志,2013,30(6):1322.
[29]Miyanohara A,Kamizato K,Juhas S,et al.Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.Mol Ther Methods Clin Dev.2016;3:16046
[30]Xu QH,Chou B,Fitzsimmons B,et al.In Vivo Gene Knockdown in Rat Dorsal Root Ganglia Mediated by Self-Complementary Adeno-Associated Virus Serotype 5 Following Intrathecal Delivery.Plos One.2012;7(3):11.
[31]Ceyzeriat K,Abjean L,Carrillo-De Sauvage MA,et al.The complex STATes of astrocyte reactivity:How are they controlled by the JAK-STAT3 pathway?.Neuroscience.2016;330:205-218.
[32]Ben Haim L,Ceyzeriat K,Carrillo-De Sauvage MA,et al.The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases.JNeurosci.2015;35(6):2817-2829.
[33]杨进顺,吕浩然,廖壮文,等.大鼠急性脊髓损伤动物模型的建立及影像学分析[J].广东医学,2011,32(5):559-561.
[34]刘小康,徐建广,连小峰,等.大鼠钳夹式急性脊髓损伤模型的制备与评价[J].中国矫形外科杂志,2012,20(14):1318-1322.
[35]杨俊松,郝定均,刘团江,等.急性脊髓损伤的临床治疗进展[J].中国脊柱脊髓杂志,2018,28(4):368-373.
[36]Sumida Y,Kamei N,Suga N,et al.The endoplasmic reticulum stress transducer old astrocyte specifically induced substance positively regulates glial scar formation in spinal cord injury.Neuroreport.2018;29(17):1443-1448.
[37]Silva NA,Sousa N,Reis RL,et al.From basics to clinical:a comprehensive review on spinal cord injury.Prog Neurobiol.2014;114:25-57.
[38]Nathan FM,Li S.Environmental cues determine the fate of astrocytes after spinal cord injury.Neural Regen Res.2017;12(12):1964-1970.
[39]Pekny M,Pekna M.Astrocyte reactivity and reactive astrogliosis:costs and benefits.Physiol Rev.2014;94(4):1077-1098.
[40]Pekny M,Wilhelmsson U,Pekna M.The dual role of astrocyte activation and reactive gliosis.Neurosci Lett.2014;565:30-38.
[41]Zou YX,Stagi M,Wang XX,et al.Gene-Silencing Screen for Mammalian Axon Regeneration Identifies Inpp5f(Sac2)as an Endogenous Suppressor of Repair after Spinal Cord Injury.JNeurosci.2015;35(29):10429-10439.
[42]Hu JL,Zhang GX,Rodemer W,et al.The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury.Neurobiol Dis.2017;98:25-35.
[2]Ahmed A,Patil AA,Agrawal DK.Immunobiology of spinal cord injuries and potential therapeutic approaches.Mol Cell Biochem.2018;441(1-2):181-189.
[3]Dalamagkas K,Tsintou M,Seifalian A,et al.Translational Regenerative Therapies for Chronic Spinal Cord Injury.Int JMol Sci.2018;19(6).pii:E1776.
[4]Ahuja CS,Nori S,Tetreault L,et al.Traumatic Spinal Cord Injury-Repair and Regeneration.Neurosurgery.2017;80(3):S9-S22.
[5]Huang X,Gu YK,Cheng XY,et al.[Astrocytes as therapeutic targets after spinal cord injury].Sheng Li Xue Bao.2017;69(6):794-804.
[6]李剑锋,闫金玉,夏润福,等.脊髓损伤胶质瘢痕形成及星形胶质细胞作用的研究与转化意义[J].中国组织工程研究,2016,20(37):5609-5616.
[7]Okada S,Hara M,Kobayakawa K,et al.Astrocyte reactivity and astrogliosis after spinal cord injury.Neurosci Res.2018;126:39-43.
[8]Sofroniew MV,Vinters HV.Astrocytes:biology and pathology.Acta Neuropathol.2010;119(1):7-35.
[9]Lu X,Xue P,Fu L,et al.HAX1 is associated with neuronal apoptosis and astrocyte proliferation after spinal cord injury.Tissue Cell.2018;54:1-9.
[10]Kameda T,Imamura T,Nakashima K.Epigenetic regulation of neural stem cell differentiation towards spinal cord regeneration.Cell Tissue Res.2018;371(1):189-199.
[11]Chen XQ,Chen C,Hao J,et al.Effect of CLIP3 Upregulation on Astrocyte Proliferation and Subsequent Glial Scar Formation in the Rat Spinal Cord via STAT3 Pathway After Injury.J Mol Neurosci.2018;64(1):117-128.
[12]Ramadan WS,Abdel-Hamid GA,Al-Karim S,et al.Neuroectodermal stem cells:A remyelinating potential in acute compressed spinal cord injury in rat model.J Biosci.2018;43(5):897-909.
[13]巩朝阳,刘开鑫,向高,等.脊髓损伤后胶质瘢痕形成的研究进展[J].中国康复理论与实践,2018,24(6):641-644.
[14]Song JL,Zheng W,Chen W,et al.Lentivirus-mediated microRNA-124 gene-modified bone marrow mesenchymal stem cell transplantation promotes the repair of spinal cord injury in rats.Exp Mol Med.201719;49(5):e332.
[15]Yin H,Shen LM,Xu C,et al.Lentivirus-Mediated Overexpression of mi R-29a Promotes Axonal Regeneration and Functional Recovery in Experimental Spinal Cord Injury via PI3K/Akt/mTORPathway.Neurochem Res.2018;43(11):2038-2046.
[16]Choudhury SR,Hudry E,Maguire CA,et al.Viral vectors for therapy of neurologic diseases.Neuropharmacology.2017;120:63-80.
[17]Saraiva J,Nobre RJ,De Almeida LP.Gene therapy for the CNSusing AAVs:The impact of systemic delivery by AAV9.JControl Release.2016;241:94-109.
[18]Gao GP,Vandenberghe LH,Alvira MR,et al.Clades of Adeno-associated viruses are widely disseminated in human tissues.J Virol.2004;78(12):6381-6388.
[19]陈如意,陈素峰,周丹,等.2型腺相关病毒作为基因治疗载体的研究进展[J].生命科学,2013,25(6):595-600.
[20]Davidson BL,Stein CS,Heth JA,et al.Recombinant adeno-associated virus type 2,4,and 5 vectors:transduction of variant cell types and regions in the mammalian central nervous system.Proc Natl Acad Sci U S A.2000;97(7):3428-3432.
[21]Murlidharan G,Samulski RJ,Asokan A.Biology of adenoassociated viral vectors in the central nervous system.Front Mol Neurosci.2014;7:76.
[22]Tadokoro T,Miyanohara A,Navarro M,et al.Subpial Adeno-associated Virus 9(AAV9)Vector Delivery in Adult Mice.J Vis Exp.2017;(125).doi:10.3791/55770.
[23]Gray SJ,Matagne V,Bachaboina L,et al.Preclinical Differences of Intravascular AAV9 Delivery to Neurons and Glia:AComparative Study of Adult Mice and Nonhuman Primates.Molecular Therapy.2011;19(6):1058-1069.
[24]Samaranch L,Salegio EA,San Sebastian W,et al.AdenoAssociated Virus Serotype 9 Transduction in the Central Nervous System of Nonhuman Primates.Human Gene Therapy.2012;23(4):382-389.
[25]Miyake N,Miyake K,Yamamoto M,et al.Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors.Brain Res.2011;1389:19-26.
[26]Povysheva T,Shmarov M,Logunov D,et al.Post-spinal cord injury astrocyte-mediated functional recovery in rats after intraspinal injection of the recombinant adenoviral vectors Ad5-VEGF and Ad5-ANG.J Neurosurg Spine.2017;27(1):105-115.
[27]李东卿.经肋间后动脉介入移植骨髓基质干细胞修复脊髓损伤的研究[D].广州:南方医科大学,2012.
[28]杞少华,王海峰,熊文平,等.骨髓间充质干细胞尾静脉注射与局部靶点注射对大鼠脊髓损伤修复效果的比较[J].中华实验外科杂志,2013,30(6):1322.
[29]Miyanohara A,Kamizato K,Juhas S,et al.Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.Mol Ther Methods Clin Dev.2016;3:16046
[30]Xu QH,Chou B,Fitzsimmons B,et al.In Vivo Gene Knockdown in Rat Dorsal Root Ganglia Mediated by Self-Complementary Adeno-Associated Virus Serotype 5 Following Intrathecal Delivery.Plos One.2012;7(3):11.
[31]Ceyzeriat K,Abjean L,Carrillo-De Sauvage MA,et al.The complex STATes of astrocyte reactivity:How are they controlled by the JAK-STAT3 pathway?.Neuroscience.2016;330:205-218.
[32]Ben Haim L,Ceyzeriat K,Carrillo-De Sauvage MA,et al.The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases.JNeurosci.2015;35(6):2817-2829.
[33]杨进顺,吕浩然,廖壮文,等.大鼠急性脊髓损伤动物模型的建立及影像学分析[J].广东医学,2011,32(5):559-561.
[34]刘小康,徐建广,连小峰,等.大鼠钳夹式急性脊髓损伤模型的制备与评价[J].中国矫形外科杂志,2012,20(14):1318-1322.
[35]杨俊松,郝定均,刘团江,等.急性脊髓损伤的临床治疗进展[J].中国脊柱脊髓杂志,2018,28(4):368-373.
[36]Sumida Y,Kamei N,Suga N,et al.The endoplasmic reticulum stress transducer old astrocyte specifically induced substance positively regulates glial scar formation in spinal cord injury.Neuroreport.2018;29(17):1443-1448.
[37]Silva NA,Sousa N,Reis RL,et al.From basics to clinical:a comprehensive review on spinal cord injury.Prog Neurobiol.2014;114:25-57.
[38]Nathan FM,Li S.Environmental cues determine the fate of astrocytes after spinal cord injury.Neural Regen Res.2017;12(12):1964-1970.
[39]Pekny M,Pekna M.Astrocyte reactivity and reactive astrogliosis:costs and benefits.Physiol Rev.2014;94(4):1077-1098.
[40]Pekny M,Wilhelmsson U,Pekna M.The dual role of astrocyte activation and reactive gliosis.Neurosci Lett.2014;565:30-38.
[41]Zou YX,Stagi M,Wang XX,et al.Gene-Silencing Screen for Mammalian Axon Regeneration Identifies Inpp5f(Sac2)as an Endogenous Suppressor of Repair after Spinal Cord Injury.JNeurosci.2015;35(29):10429-10439.
[42]Hu JL,Zhang GX,Rodemer W,et al.The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury.Neurobiol Dis.2017;98:25-35.