围产期短暂性缺氧NeuroD变化与神经再生相关性研究
摘要
目的
观察短暂性缺氧后鼠脑神经源性分化因子(Neurogenic differentiation, NeuroD)表达量的变化及其与神经系统再生的关系,探讨其在神经系统再生中的可能作用。
方法
通过RT-PCR检测短暂性缺氧后NeuroD mRNA表达量的变化。进而通过体外培养大鼠海马神经元,RT-PCR检测细胞缺氧后NeuroD mRNA表达量的变化,BrdU免疫组化检测缺氧后细胞增殖情况;通过“延迟剖宫产术”建立胎鼠宫内窘迫模型,对模型组动物进行神经行为学检测,原位杂交技术检测术后不同天数海马结构NeuroD mRNA表达量的变化。
结果
短暂性缺氧后NeuroD mRNA表达量增高,并且表达量随着缺氧时间的延长而不断增高。免疫荧光结果显示,在大脑皮质及皮质下区域NeuroD呈点状或条索状弥散分布,窘迫前后NeuroD表达量未见明显变化(P>0.05)。在尾壳核区域NeuroD的表达相对集中,窘迫后表达量明显增高。体外培养5d的海马神经元缺氧3h后RT-PCR检测到NeuroD mRNA表达量明显增高,BrdU免疫组化结果显示缺氧再培养后检测到BrdU阳性细胞,即有的神经元返回细胞周期,重新进行分裂增殖。在体通过“延迟剖宫产术”建立宫内窘迫模型。大鼠神经行为学检测结果表明,宫内窘迫10min组和对照组相比,大鼠的动作协调能力和学习能力统计学上未见明显差异,但模型组大鼠的重拾记忆能力有明显提高(P< 0.05)。原位杂交结果显示,模型组无论在海马结构的齿状回颗粒下层还是CA1区,NeuroD在P13、P20、P27天的表达量与对照组相比都有明显增高,且在P20天最明显。
结论
1.在体动物实验证明短暂性缺氧后NeuroD表达量增高
2.体外培养海马神经元缺氧后NeuroD表达量增高,与之伴随的是有的神经元返回细胞周期,重新进行分裂增殖
3.海马齿状回颗粒下层和CA1区NeuroD表达量的变化与神经系统再生具有相关性
以上表明,高表达的NeuroD可能参与了神经干细胞的分化,在神经系统再生过程中发挥了一定作用
Objective To observe the influence of transient hypoxia on the expression of NeuroD in rat brain and investigate its possible roles in neural regeneration.
Methods To investigate the influence of transient hypoxia on the expression of NeuroD, asphyxia was induced in rat pups by performing a delayed cesarean section on pregnant Sprague-Dawley rats.The expression of NeuroD was measured by RT-PCR and immunofluorescence. To investigate the role of NeuroD in neural regeneration, the delayed cesarean section was performed on pregnant SD rats and the newborn rats were returned to dams. In vitro, influence of transient hypoxia on the expression of NeuroD was analyzed on the outcome of embryonic rat neurons in culture. The expression of NeuroD was measured by RT-PCR and the cell proliferation was measured by incorporation of BrdU. In vivo, neurobehavioral consequences of the hypoxic episode were evaluated by using standard behavioral tests for psychomotor and coordination performances and learning capacities.The expression of NeuroD was monitored at the level of DG and CA1 layer of the hippocampus by in situ hybridization.
Results NeuroD mRNA increased after the exposure to hypoxia ( P < 0.05 ).Compared with the control group, NeuroD showed no much difference at cerebral cortex but increased significantly at nucleus caudatus putamen detected by immunofluorescence. As revealed by neurobehavioral consequences, hypoxia did not impair psychomotor or learning capacities but improved memory retrieval scores. In vitro, following hypoxia for 3 hours in cultured neurons, NeuroD increased distinctly and with incorporation of BrdU revealed an accumulation of proliferating cells. Following hypoxia in vivo induced higher expression of NeuroD in DG and CA1 subfield of the hippocampus at days 13、20、27 post hypoxia and significantly at P20.
Conclusions First, the expression of NeuroD was increased the exposure to hypoxia. Second, the expression of NeuroD was increased post asphyxia in cultured neurons, following with cell proliferation. Third, the differential expression of NeuroD coincided with the dalyed brain neurogenesis. As mentioned above, NeuroD seems to play a role in the process of neurogenesis.
观察短暂性缺氧后鼠脑神经源性分化因子(Neurogenic differentiation, NeuroD)表达量的变化及其与神经系统再生的关系,探讨其在神经系统再生中的可能作用。
方法
通过RT-PCR检测短暂性缺氧后NeuroD mRNA表达量的变化。进而通过体外培养大鼠海马神经元,RT-PCR检测细胞缺氧后NeuroD mRNA表达量的变化,BrdU免疫组化检测缺氧后细胞增殖情况;通过“延迟剖宫产术”建立胎鼠宫内窘迫模型,对模型组动物进行神经行为学检测,原位杂交技术检测术后不同天数海马结构NeuroD mRNA表达量的变化。
结果
短暂性缺氧后NeuroD mRNA表达量增高,并且表达量随着缺氧时间的延长而不断增高。免疫荧光结果显示,在大脑皮质及皮质下区域NeuroD呈点状或条索状弥散分布,窘迫前后NeuroD表达量未见明显变化(P>0.05)。在尾壳核区域NeuroD的表达相对集中,窘迫后表达量明显增高。体外培养5d的海马神经元缺氧3h后RT-PCR检测到NeuroD mRNA表达量明显增高,BrdU免疫组化结果显示缺氧再培养后检测到BrdU阳性细胞,即有的神经元返回细胞周期,重新进行分裂增殖。在体通过“延迟剖宫产术”建立宫内窘迫模型。大鼠神经行为学检测结果表明,宫内窘迫10min组和对照组相比,大鼠的动作协调能力和学习能力统计学上未见明显差异,但模型组大鼠的重拾记忆能力有明显提高(P< 0.05)。原位杂交结果显示,模型组无论在海马结构的齿状回颗粒下层还是CA1区,NeuroD在P13、P20、P27天的表达量与对照组相比都有明显增高,且在P20天最明显。
结论
1.在体动物实验证明短暂性缺氧后NeuroD表达量增高
2.体外培养海马神经元缺氧后NeuroD表达量增高,与之伴随的是有的神经元返回细胞周期,重新进行分裂增殖
3.海马齿状回颗粒下层和CA1区NeuroD表达量的变化与神经系统再生具有相关性
以上表明,高表达的NeuroD可能参与了神经干细胞的分化,在神经系统再生过程中发挥了一定作用
Objective To observe the influence of transient hypoxia on the expression of NeuroD in rat brain and investigate its possible roles in neural regeneration.
Methods To investigate the influence of transient hypoxia on the expression of NeuroD, asphyxia was induced in rat pups by performing a delayed cesarean section on pregnant Sprague-Dawley rats.The expression of NeuroD was measured by RT-PCR and immunofluorescence. To investigate the role of NeuroD in neural regeneration, the delayed cesarean section was performed on pregnant SD rats and the newborn rats were returned to dams. In vitro, influence of transient hypoxia on the expression of NeuroD was analyzed on the outcome of embryonic rat neurons in culture. The expression of NeuroD was measured by RT-PCR and the cell proliferation was measured by incorporation of BrdU. In vivo, neurobehavioral consequences of the hypoxic episode were evaluated by using standard behavioral tests for psychomotor and coordination performances and learning capacities.The expression of NeuroD was monitored at the level of DG and CA1 layer of the hippocampus by in situ hybridization.
Results NeuroD mRNA increased after the exposure to hypoxia ( P < 0.05 ).Compared with the control group, NeuroD showed no much difference at cerebral cortex but increased significantly at nucleus caudatus putamen detected by immunofluorescence. As revealed by neurobehavioral consequences, hypoxia did not impair psychomotor or learning capacities but improved memory retrieval scores. In vitro, following hypoxia for 3 hours in cultured neurons, NeuroD increased distinctly and with incorporation of BrdU revealed an accumulation of proliferating cells. Following hypoxia in vivo induced higher expression of NeuroD in DG and CA1 subfield of the hippocampus at days 13、20、27 post hypoxia and significantly at P20.
Conclusions First, the expression of NeuroD was increased the exposure to hypoxia. Second, the expression of NeuroD was increased post asphyxia in cultured neurons, following with cell proliferation. Third, the differential expression of NeuroD coincided with the dalyed brain neurogenesis. As mentioned above, NeuroD seems to play a role in the process of neurogenesis.
引文
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4. Bruel-Junqerman E, Rampon C, Laroche S. Adult hippocampal neurogenesis, synaptic plasticity and memory: facts and hypotheses. Rev Neurosci. 2007,18: 93-114.
5. Amrein I, Lipp HP. Adult hippocampal neurogenesis of mammals: evolution and life history. Biol Lett.2009,5:141-144.
6. Lee JE. Basic helix-loop-helix genes in neural development. Curr Opin Neurobiol. 1997,7:13?20.
7. Lee JE. NeuroD and neurogenesis. Developmental Neuroscience.1997,19:27-32.
8. Bray SJ. Expression and function of Enhancer of split bHLH proteins during Drosophila neurogenesis. Perspect Dev Neurobiol.1997,4:313-323.
9. Fisher A, Caudy M. The function of hairy-related bHLH repressor proteins in cell fate decisions.Bioessays.1998, 20:298-306.
10. Lee JE, Hollenberg SM, Snider L, et al. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein.Science.1995,268, 836?844.
11. Takahashi J, Palmer TD, Gage FH. Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures. J Neurobiol. 1999,38:65?81.
12. Shin MH, Lee EG, Lee SH, et al. Neural cell adhesion molecule (NCAM) promotes the differentiation of hippocampal precursor cells to a neuronal lineage, especially to a glutamatergic neural cell type. Exp Mol Med. 2002,34:401?410.
13. Chris K. Neurogenesis in embryos and in adult neural stem cell.Neurosci.2002, 22:639?643.
14. Roybon L, Deierborg T, Brundin P, et al. Involvement of Ngn2, Tbr and NeuroD proteins during postnatal olfactory bulb neurogenesis. Eur J Neurosci. 2009, 29: 232 -243.
15. Wang Y, Chen KP, Yao Q. [Progress of studies on bHLH transcription factor families].Yi Chuan.2008,30(7):821-830.
16. Stevens JD, Roalson EH, Skinner MK. Phylogenetic and expression analysis of the basic helix-loop-helix transcription factor gene family: genomic approach to cellular differentiation. Differentiation. 2008,76:1006-1022.
17. Wullimann MF, Rink E, Vernier P, et al. Secondary neurogenesis in the brain of the African clawed frog, Xenopus laevis, as revealed by PCNA, Delta-1, Neurogenin-related-1, and NeuroD expression. Journal of Comparative Neurology. 2005,489:387-402.
18. Pourie G,Blaise S,Trabalon M,et al. Mild, non-lesioning transient hypoxia in the newborn rat induces delayed brain neurogenesis associated with improved memory scores. J Neurosci.2006,140: 1369-1379.
19. Vert P, Daval JL. [Cell death and neurogenesis after hypoxia: a brain repair mechanism in the developing rat?]. Bull Acad Natl Med.2006,190:469-81; discussion 481-484.
20. Khokhlova VA, Kazakova PB. Effect of maternal hypoxia on the neurogenesis of the cerebral cortex of rat progeny. Bull Eksp Biol Med.1979,87:485–487.
21. Bjelke B, Andersson K, Ogren SO , et al.Asphyctic lesion: proliferation of tyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigra and functional changes in dopamine neurotransmission. Brain Res.1991, 543:1-9.
22. Ishizuka N, Minami K, Okumachi A, et al. Induction by NeuroD of the components required for regulated exocytosis. Biochem Biophys Res Commun. 2007,354:271-277.
23. Gidday JM, Fitzgibbons JC, Shah AR, Park TS. Neuroprotection from ischemic brain injury by hypoxic preconditioning in the neonatal rat. Neurosci Lett.1994,168:221–224.
24. Daval JL,Vert P. Apoptosis and neurogenesis after transient hypoxia in the developing rat brain. Semin Perinatol. 2004,28:257-263.
25. Stattford A, Wheatherhall JAC. The survival of young rats in nitrogen. J Physiol. 1960,153:457-472.
26. Haddad GG, Donelly DF. O2 deprivation induces a major depolarization in brainstem neurons in the adult but not in the neonate. J Physiol Lond.1990, 429: 411-428.
27. Duffy TE, Kohle SJ, Vannucci RC. Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J Neurochem.1975,24:271–276.
28. Gould E, Reeves AJ, Fallah M, et al. Hippocampal neurogenesis in adult Old World primates. Proc Natl Acad Sci USA.1999, 96: 5263–5267.
29. Gould E, Vail N, Wagers M, et al. Adult-generated hippocampal and neocortical neurons in macaques have a transient existence. Proc Natl Acad Sci USA.2001,98: 10910–10917.
30. Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al.Neurogenesis in the adult human hippocampus. Nat Med.1998,4: 1313–1317.
31. Bossenmeyer C, Chihab R, Muller S, et al. Hypoxia/reoxygenation induces apoptosis through biphasic induction of protein synthesis in central neurons. Brain Res.1998,787:107-116.
32. Bossenmeyer-Pourie′C, Chihab R, Schroeder H, et al. Transient hypoxia may lead to neuronal proliferation in the developing mammalian brain: From apoptosis to cell cycle completion. Neuroscience.1999,91:221-231.
33. Elliott RC, Khademi S, Pleasure SJ, et al. Differential regulation of basic helix-loop-helix mRNAs in the dentate gyrus following status epilepticus. Neuroscience.2001,106:79?88.
34. Jin K, Peel AL, Mao XO, et al. Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci USA. 2004,101:343?347.
35. Kawai T, Takagi N, Miyake-Takagi K, et al. Characterization of BrdU-positive neurons induced by transient global ischemia in adult hippocampus. Cereb. Blood Flow Metab. 2004,24:548?555.
36. Pleasure SJ, Collins AE, Lowenstein DH. Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development. J Neurosci. 2000, 20:6095?6105.
37. Uittenbogaard M, Chiaramello A. Differential expression patterns of the basic helix-loop-helix transcription factors during aging of the murine brain. Neurosci. Lett. 2000,280:95?98.
1. McKay R. Stem cells in the central nervous system.Science.1997,276:66-77.
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12. Takahashi J, Palmer TD, Gage FH. Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures. J Neurobiol.1999, 38:65-81.
13. Shin MH, Lee EG, Lee SH,et al.Neural cell adhesion molecule (NCAM) promotes the differentiation of hippocampal precursor cells to a neuronal lineage, especially to a glutamatergic neural cell type. Exp Mol Med. 2002,34:401-410.
14. Ochocinska MJ, Hitchcock PF. NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish. Mech Dev. 2009,126:128-141.
15. Elliott RC, Khademi S, Pleasure SJ, et al. Differential regulation of basic helix-loop-helix mRNAs in the dentate gyrus following status epilepticus. Neuroscience.2001,106:79-88.
16. Kawai T, Takagi N, Miyake-Takagi K, et al. Characterization of BrdU-positive neurons induced by transient global ischemia in adult hippocampus. Cereb Blood Flow Metab.2004,24:548-555.
17. Pleasure SJ, Collins AE, Lowenstein DH. Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development. J Neurosci. 2000,20:6095-6105.
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2. Shapiro EM,Gonzalez-Percz O, Manuel García-Verdugo J ,et a1. Magnetic resonance imaging of the migration of neuronal precursors generated in the adult rodent brain.Neuroimage.2006,32:1150-1157.
3. Ehninqer D,Kempermann G. Neurogenesis in the adult hippocampus. Cell Tissue Res. 2008,331:243-50.
4. Bruel-Junqerman E, Rampon C, Laroche S. Adult hippocampal neurogenesis, synaptic plasticity and memory: facts and hypotheses. Rev Neurosci. 2007,18: 93-114.
5. Amrein I, Lipp HP. Adult hippocampal neurogenesis of mammals: evolution and life history. Biol Lett.2009,5:141-144.
6. Lee JE. Basic helix-loop-helix genes in neural development. Curr Opin Neurobiol. 1997,7:13?20.
7. Lee JE. NeuroD and neurogenesis. Developmental Neuroscience.1997,19:27-32.
8. Bray SJ. Expression and function of Enhancer of split bHLH proteins during Drosophila neurogenesis. Perspect Dev Neurobiol.1997,4:313-323.
9. Fisher A, Caudy M. The function of hairy-related bHLH repressor proteins in cell fate decisions.Bioessays.1998, 20:298-306.
10. Lee JE, Hollenberg SM, Snider L, et al. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein.Science.1995,268, 836?844.
11. Takahashi J, Palmer TD, Gage FH. Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures. J Neurobiol. 1999,38:65?81.
12. Shin MH, Lee EG, Lee SH, et al. Neural cell adhesion molecule (NCAM) promotes the differentiation of hippocampal precursor cells to a neuronal lineage, especially to a glutamatergic neural cell type. Exp Mol Med. 2002,34:401?410.
13. Chris K. Neurogenesis in embryos and in adult neural stem cell.Neurosci.2002, 22:639?643.
14. Roybon L, Deierborg T, Brundin P, et al. Involvement of Ngn2, Tbr and NeuroD proteins during postnatal olfactory bulb neurogenesis. Eur J Neurosci. 2009, 29: 232 -243.
15. Wang Y, Chen KP, Yao Q. [Progress of studies on bHLH transcription factor families].Yi Chuan.2008,30(7):821-830.
16. Stevens JD, Roalson EH, Skinner MK. Phylogenetic and expression analysis of the basic helix-loop-helix transcription factor gene family: genomic approach to cellular differentiation. Differentiation. 2008,76:1006-1022.
17. Wullimann MF, Rink E, Vernier P, et al. Secondary neurogenesis in the brain of the African clawed frog, Xenopus laevis, as revealed by PCNA, Delta-1, Neurogenin-related-1, and NeuroD expression. Journal of Comparative Neurology. 2005,489:387-402.
18. Pourie G,Blaise S,Trabalon M,et al. Mild, non-lesioning transient hypoxia in the newborn rat induces delayed brain neurogenesis associated with improved memory scores. J Neurosci.2006,140: 1369-1379.
19. Vert P, Daval JL. [Cell death and neurogenesis after hypoxia: a brain repair mechanism in the developing rat?]. Bull Acad Natl Med.2006,190:469-81; discussion 481-484.
20. Khokhlova VA, Kazakova PB. Effect of maternal hypoxia on the neurogenesis of the cerebral cortex of rat progeny. Bull Eksp Biol Med.1979,87:485–487.
21. Bjelke B, Andersson K, Ogren SO , et al.Asphyctic lesion: proliferation of tyrosine hydroxylase-immunoreactive nerve cell bodies in the rat substantia nigra and functional changes in dopamine neurotransmission. Brain Res.1991, 543:1-9.
22. Ishizuka N, Minami K, Okumachi A, et al. Induction by NeuroD of the components required for regulated exocytosis. Biochem Biophys Res Commun. 2007,354:271-277.
23. Gidday JM, Fitzgibbons JC, Shah AR, Park TS. Neuroprotection from ischemic brain injury by hypoxic preconditioning in the neonatal rat. Neurosci Lett.1994,168:221–224.
24. Daval JL,Vert P. Apoptosis and neurogenesis after transient hypoxia in the developing rat brain. Semin Perinatol. 2004,28:257-263.
25. Stattford A, Wheatherhall JAC. The survival of young rats in nitrogen. J Physiol. 1960,153:457-472.
26. Haddad GG, Donelly DF. O2 deprivation induces a major depolarization in brainstem neurons in the adult but not in the neonate. J Physiol Lond.1990, 429: 411-428.
27. Duffy TE, Kohle SJ, Vannucci RC. Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J Neurochem.1975,24:271–276.
28. Gould E, Reeves AJ, Fallah M, et al. Hippocampal neurogenesis in adult Old World primates. Proc Natl Acad Sci USA.1999, 96: 5263–5267.
29. Gould E, Vail N, Wagers M, et al. Adult-generated hippocampal and neocortical neurons in macaques have a transient existence. Proc Natl Acad Sci USA.2001,98: 10910–10917.
30. Eriksson PS, Perfilieva E, Bjork-Eriksson T, et al.Neurogenesis in the adult human hippocampus. Nat Med.1998,4: 1313–1317.
31. Bossenmeyer C, Chihab R, Muller S, et al. Hypoxia/reoxygenation induces apoptosis through biphasic induction of protein synthesis in central neurons. Brain Res.1998,787:107-116.
32. Bossenmeyer-Pourie′C, Chihab R, Schroeder H, et al. Transient hypoxia may lead to neuronal proliferation in the developing mammalian brain: From apoptosis to cell cycle completion. Neuroscience.1999,91:221-231.
33. Elliott RC, Khademi S, Pleasure SJ, et al. Differential regulation of basic helix-loop-helix mRNAs in the dentate gyrus following status epilepticus. Neuroscience.2001,106:79?88.
34. Jin K, Peel AL, Mao XO, et al. Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci USA. 2004,101:343?347.
35. Kawai T, Takagi N, Miyake-Takagi K, et al. Characterization of BrdU-positive neurons induced by transient global ischemia in adult hippocampus. Cereb. Blood Flow Metab. 2004,24:548?555.
36. Pleasure SJ, Collins AE, Lowenstein DH. Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development. J Neurosci. 2000, 20:6095?6105.
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