6%和8%缺氧对新生大鼠脑白质损伤的差异及其机制
|
摘要
目的:观察新生大鼠不同缺血缺氧(hypoxia-ischemia,HI)条件下脑白质的损伤状况,并探其损伤的机制。方法:新生3 d SD大鼠随机分为正常对照组、6%缺氧和8%缺氧损伤模型组,模型组大鼠进行右侧颈总动脉结扎,2 h后进行6%或8%缺氧,建立HI脑白质损伤动物模型。分别于2周、3周和4周取脑制作冰冻切片,免疫荧光检测髓鞘碱性蛋白(myelin basic protein,MBP)和髓鞘相关糖蛋白(myelin-associated glycoprotein,MAG)的变化,RT-PCR检测增殖和分化相关因子硫酸软骨素蛋白多糖4(chrondroitin sulfate proteoglycan 4,CSPG4)和类胰岛素一号增长因子(insulin-like growth factors-1,IGF-1)的表达,探索HI脑白质损伤的机制。结果:体重统计显示:与正常对照组大鼠相比,HI损伤后1周、2周、3周和4周时模型大鼠平均体重在各时间点均显著性降低;6%和8%模型大鼠平均体重在各时间点无显著差异。与对侧MBP和MAG荧光强度相比,3周和4周时6%缺氧组损伤侧MBP和MAG荧光强度的比值均较8%缺氧组减少(P<0.05)。RT-PCR结果显示:同一时间点,6%缺氧组CSPG4的表达显著高于8%缺氧组(P<0.05),而IGF-1的表达显著低于8%缺氧组(P<0.05)。结论:6%或8%缺氧损伤均可造成新生大鼠明显的脑白质损伤,但6%氧损伤导致大鼠脑白质损伤较8%缺氧加重,其机制可能与不同缺氧程度导致的少突胶质前体细胞分化抑制因子CSPG4和增殖分化因子IGF-1的不同表达有关。
Objective: To explore the degree and mechanism of cerebral white matter damage in neonatal rats under different conditions of hypoxia-ischemia( HI) injury. Methods: 3-day-old neonatal SD rats were randomly divided into3 groups: control,6% hypoxia and 8% hypoxia groups. Right common carotid artery ligation was carried out to create the HI injury model,and then 6% or 8% oxygen was applied to perform hypoxia experiments for 2 hours. Immunofluorescence staining of myelin basic protein( MBP) and myelin-associated glycoprotein( MAG) were applied to assess the changes of mature oligodendrocytes in different hypoxia conditions. Also,RT-PCR was carried out to detect the expression of chrondroitin sulfate proteoglycan 4( CSPG4) and insulin-like growth factors-1( IGF-1). Results: Compared with the control group rats,the average body weight was markedly reduced in HI injury rats at 1 week,2 weeks,3 weeks and 4 weeks,while no notable difference was found in the average body weight between 6% and 8% groups at each time point. When compared with the contralateral corpus callosum,the fluorescence intensity of MBP and MAG in6% hypoxia group was lower than that in 8% hypoxia group in ipsilateral corpus callosum at 3 weeks and 4 weeks( P <0. 05). The results of RT-PCR displayed that when compared with 8% hypoxia group,the expression of GSPG4 was dramatically enhanced in 6% hypoxia group. In contrast,the level of IGF-1 was obviously lower in 6% hypoxia group than that in the 8% hypoxia group. Conclusion: Both 6% and 8% oxygen cause damage to white matter of neonatal rats obviously,but 6% oxygen can lead to a more severe injury than 8% oxygen. The possible mechanisms may be related to different hypoxia induced the changes of CSPG4 and IGF-1 in oligodendrocyte precursor cells.
Objective: To explore the degree and mechanism of cerebral white matter damage in neonatal rats under different conditions of hypoxia-ischemia( HI) injury. Methods: 3-day-old neonatal SD rats were randomly divided into3 groups: control,6% hypoxia and 8% hypoxia groups. Right common carotid artery ligation was carried out to create the HI injury model,and then 6% or 8% oxygen was applied to perform hypoxia experiments for 2 hours. Immunofluorescence staining of myelin basic protein( MBP) and myelin-associated glycoprotein( MAG) were applied to assess the changes of mature oligodendrocytes in different hypoxia conditions. Also,RT-PCR was carried out to detect the expression of chrondroitin sulfate proteoglycan 4( CSPG4) and insulin-like growth factors-1( IGF-1). Results: Compared with the control group rats,the average body weight was markedly reduced in HI injury rats at 1 week,2 weeks,3 weeks and 4 weeks,while no notable difference was found in the average body weight between 6% and 8% groups at each time point. When compared with the contralateral corpus callosum,the fluorescence intensity of MBP and MAG in6% hypoxia group was lower than that in 8% hypoxia group in ipsilateral corpus callosum at 3 weeks and 4 weeks( P <0. 05). The results of RT-PCR displayed that when compared with 8% hypoxia group,the expression of GSPG4 was dramatically enhanced in 6% hypoxia group. In contrast,the level of IGF-1 was obviously lower in 6% hypoxia group than that in the 8% hypoxia group. Conclusion: Both 6% and 8% oxygen cause damage to white matter of neonatal rats obviously,but 6% oxygen can lead to a more severe injury than 8% oxygen. The possible mechanisms may be related to different hypoxia induced the changes of CSPG4 and IGF-1 in oligodendrocyte precursor cells.
引文
[1]Lee YA.White matter injury of prematurity:Its mechanisms and clinical features[J].J Pathol Transl Med,2017,51:449-455.
[2]Gano D.White matter injury in premature newborns[J].Neonatal Netw,2016,35:73-77.
[3]Koo E,Sheldon RA,Lee BS,et al.Effects of therapeutic hypothermia on white matter injury from murine neonatal hypoxia-ischemia[J].Pediatr Res,2017,82:518-526.
[4]Gonzales-Portillo GS,Reyes S,Aguirre D,et al.Stem cell therapy for neonatal hypoxic-ischemic encephalopathy[J].Front Neurol,2014,5:147.
[5]Yasuhara T,Matsukawa N,Yu G,et al.Behavioral and histological characterization of intrahippocampal grafts of human bone marrowderived multipotent progenitor cells in neonatal rats with hypoxic-ischemic injury[J].Cell Transplant,2006,15:231-238.
[6]Tuor UI,Morgunov M,Sule M,et al.Cellular correlates of longitudinal diffusion tensor imaging of axonal degeneration following hypoxic-ischemic cerebral infarction in neonatal rats[J].Neuroimage Clin,2014,6:32-42.
[7]van de Looij Y,Chatagner A,Quairiaux C,et al.Multi-modal assessment of long-term erythropoietin treatment after neonatal hypoxic-ischemic injury in rat brain[J].PLo S One,2014,9:e95643.
[8]Chen LX,Ma SM,Zhang P,et al.Neuroprotective effects of oligodendrocyte progenitor cell transplantation in premature rat brain following hypoxic-ischemic injury[J].PLo S One,2015,10:e115997.
[9]van Velthoven CT,Dzietko M,Wendland MF,et al.Mesenchymal stem cells attenuate MRI-identifiable injury,protect white matter,and improve long-term functional outcomes after neonatal focal stroke in rats[J].J Neurosci Res.2017,95:1225-1236.
[10]Rosen CL,Bunge RP,Ard MD,et al.Type 1 astrocytes inhibit myelination by adult rat oligodendrocytes in vitro[J].J Neurosci,1989,9:3371-3379.
[11]Brambilla R,Morton PD,Ashbaugh JJ,et al.Astrocytes play a key role in EAE pathophysiology by orchestrating in the CNS the inflammatory response of resident and peripheral immune cells and by suppressing remyelination[J].Glia,2014,62:452-467.
[12]Guo AC,Chu T,Liu XQ,et al.Reactivated astrocytes as a possible source of oligodendrocyte precursors for remyelination in remitting phase of experimental autoimmune encephalomyelitis rats[J].Am J Transl Res.2016;8:5637-5645.
[13]Alizadeh A and Karimi-Abdolrezaee S.Microenvironmental regulation of oligodendrocyte replacement and remyelination in spinal cord injury[J].J Physiol,2016,594:3539-3552.
[14]Dyck SM and Karimi-Abdolrezaee S.Chondroitin sulfate proteoglycans:Key modulators in the developing and pathologic central nervous system[J].Exp Neurol,2015,269:169-187.
[15]Cua RC,Lau LW,Keough MB,et al.Overcoming neurite-inhibitory chondroitin sulfate proteoglycans in the astrocyte matrix[J].Glia,2013,61:972-984.
[16]Dyck SM,Alizadeh A,Santhosh KT,et al.Chondroitin sulfate proteoglycans negatively modulate spinal cord neural precursor cells by signaling through LAR and RPTP sigma and modulation of the Rho/ROCK pathway[J].Stem Cells,2015,33:2550-2563.
[17]Lau LW,Keough MB,Haylock-Jacobs S,et al.Chondroitin sulfate proteoglycans in demyelinated lesions impair remyelination[J].Ann Neurol,2012,72:419-432.
[18]Beck KD,Powell-Braxton L,Widmer HR,et al.Igf1 gene disruption results in reduced brain size,CNS hypomyelination,and loss of hippocampal granule and striatal parvalbumin-containing neurons[J].Neuron,1995,14:717-730.
[19]Lioutas VA,Alfaro-Martinez F,Bedoya F,et al.Intranasal insulin and insulin-like growth factor 1 as neuroprotectants in acute ischemic stroke[J].Transl Stroke Res,2015,6:264-275.
[20]Hlavica M,Delparente A,Good A,et al.Intrathecal insulin-like growth factor 1 but not insulin enhances myelin repair in young and aged rats[J].Neurosci Lett,2017,648:41-46.
[2]Gano D.White matter injury in premature newborns[J].Neonatal Netw,2016,35:73-77.
[3]Koo E,Sheldon RA,Lee BS,et al.Effects of therapeutic hypothermia on white matter injury from murine neonatal hypoxia-ischemia[J].Pediatr Res,2017,82:518-526.
[4]Gonzales-Portillo GS,Reyes S,Aguirre D,et al.Stem cell therapy for neonatal hypoxic-ischemic encephalopathy[J].Front Neurol,2014,5:147.
[5]Yasuhara T,Matsukawa N,Yu G,et al.Behavioral and histological characterization of intrahippocampal grafts of human bone marrowderived multipotent progenitor cells in neonatal rats with hypoxic-ischemic injury[J].Cell Transplant,2006,15:231-238.
[6]Tuor UI,Morgunov M,Sule M,et al.Cellular correlates of longitudinal diffusion tensor imaging of axonal degeneration following hypoxic-ischemic cerebral infarction in neonatal rats[J].Neuroimage Clin,2014,6:32-42.
[7]van de Looij Y,Chatagner A,Quairiaux C,et al.Multi-modal assessment of long-term erythropoietin treatment after neonatal hypoxic-ischemic injury in rat brain[J].PLo S One,2014,9:e95643.
[8]Chen LX,Ma SM,Zhang P,et al.Neuroprotective effects of oligodendrocyte progenitor cell transplantation in premature rat brain following hypoxic-ischemic injury[J].PLo S One,2015,10:e115997.
[9]van Velthoven CT,Dzietko M,Wendland MF,et al.Mesenchymal stem cells attenuate MRI-identifiable injury,protect white matter,and improve long-term functional outcomes after neonatal focal stroke in rats[J].J Neurosci Res.2017,95:1225-1236.
[10]Rosen CL,Bunge RP,Ard MD,et al.Type 1 astrocytes inhibit myelination by adult rat oligodendrocytes in vitro[J].J Neurosci,1989,9:3371-3379.
[11]Brambilla R,Morton PD,Ashbaugh JJ,et al.Astrocytes play a key role in EAE pathophysiology by orchestrating in the CNS the inflammatory response of resident and peripheral immune cells and by suppressing remyelination[J].Glia,2014,62:452-467.
[12]Guo AC,Chu T,Liu XQ,et al.Reactivated astrocytes as a possible source of oligodendrocyte precursors for remyelination in remitting phase of experimental autoimmune encephalomyelitis rats[J].Am J Transl Res.2016;8:5637-5645.
[13]Alizadeh A and Karimi-Abdolrezaee S.Microenvironmental regulation of oligodendrocyte replacement and remyelination in spinal cord injury[J].J Physiol,2016,594:3539-3552.
[14]Dyck SM and Karimi-Abdolrezaee S.Chondroitin sulfate proteoglycans:Key modulators in the developing and pathologic central nervous system[J].Exp Neurol,2015,269:169-187.
[15]Cua RC,Lau LW,Keough MB,et al.Overcoming neurite-inhibitory chondroitin sulfate proteoglycans in the astrocyte matrix[J].Glia,2013,61:972-984.
[16]Dyck SM,Alizadeh A,Santhosh KT,et al.Chondroitin sulfate proteoglycans negatively modulate spinal cord neural precursor cells by signaling through LAR and RPTP sigma and modulation of the Rho/ROCK pathway[J].Stem Cells,2015,33:2550-2563.
[17]Lau LW,Keough MB,Haylock-Jacobs S,et al.Chondroitin sulfate proteoglycans in demyelinated lesions impair remyelination[J].Ann Neurol,2012,72:419-432.
[18]Beck KD,Powell-Braxton L,Widmer HR,et al.Igf1 gene disruption results in reduced brain size,CNS hypomyelination,and loss of hippocampal granule and striatal parvalbumin-containing neurons[J].Neuron,1995,14:717-730.
[19]Lioutas VA,Alfaro-Martinez F,Bedoya F,et al.Intranasal insulin and insulin-like growth factor 1 as neuroprotectants in acute ischemic stroke[J].Transl Stroke Res,2015,6:264-275.
[20]Hlavica M,Delparente A,Good A,et al.Intrathecal insulin-like growth factor 1 but not insulin enhances myelin repair in young and aged rats[J].Neurosci Lett,2017,648:41-46.