神经机械性损伤中脑红蛋白表达及作用的研究
摘要
氧是生命活动的基本物质之一,而氧的代谢和利用一直是生命科学研究中的活跃领域。众所周知,人体内存在两种与氧密切相关的球蛋白:血红蛋白(hemoglobin,HGB)和肌红蛋白(myoglobin,MGB)。它们是机体实现氧的储存、利用、转运、线粒体有氧活动及活性氧的清除等过程中的重要载体。成人脑虽然仪占体重的2%,但其有氧代谢非常活跃,耗氧量占人体总耗氧量的20%左右。那么神经系统中是否存在一种特异性的携氧蛋白以促进脑的氧和能量代谢呢?这一问题激起了科研工作者的广泛兴趣。2000年Burmester~[1]等首次报道,脊椎动物神经系统内存在第三种单体携氧珠蛋白——脑红蛋白(neuroglobin,NGB),它住皮层神经元及视网膜中呈高表达,提示其可能参予了神经系统氧与能量的代谢活动,为神经系统氧利用的研究带来新的曙光。已有的研究结果显示~[2-6]:缺氧可以诱导NGB表达上调,同时,高表达NGB有助于神经组织耐受缺氧损伤,而抑制NGB表达则会加重其损伤程度。以上研究为NGB作为内源性神经保护因子提供了论据,为治疗缺氧性神经损伤提供了新思路。
在神经外科的临床工作中,颅脑创伤是最常见的病种之一。创伤后脑组织水肿,对氧的摄取和利用明显下降是引起脑损伤的重要因素。而NGB作为神经系统特有的携氧球蛋白,研究其在神经系统机械性损伤中的表达和作用,将为临床工作中对颅脑创伤的诊断、治疗提供新的契机和切入点。本研究包括:
1、建立体外培养原代神经元机械性损伤模型:手术取胎鼠人脑皮层,分离纯化后作原代神经元培养:用抗神元特异性蛋白——神经丝蛋白(Neurofilament protein,NF)抗体鉴定细胞纯度;对Banker体外损伤模型进行改良,采用自行设计的模版对培养神经元进行机械性损伤,并在损伤后行光镜下观察及免疫组化检测c-Fos蛋白来证实神经元的损伤。
2、分别采用免疫组织化学、Western Blot技术检测神经元损伤后NGB的表达变化:神经元NGB染色采用链霉素抗生物素-过氧化物酶连接法(SP法),并采用图像分析技术(Image J软件,美国国立卫生院--NIH)观测损伤前、后不同时间点神经元的NGB免疫组化反应阳性信号的灰度值;细胞裂解提取蛋白后,采用Western Blot技术检测神经元损伤前、后各时间点NGB的表达。两种检测方法的结果一致。
3、增加NGB的表达对损伤神经源性细胞活性的影响:因神经元的转染率低,因而采用转染率高的神经源性细胞(B104细胞)进行实验。先按照损伤模型对B104细胞进行机械性损伤,采用Western Blot技术检测B104细胞损伤后NGB的表达变化,确定其变化趋势与原代神经元相同;然后采用军事医学科学院二所十三室制备的pCMV-Myc-NGB质粒转染B104细胞,用Western Blot检测技术验证B104细胞转染pCMV-Myc-NGB质粒后NGB表达增多:选取损伤后0.5h、2h两个时间点,分别测定并比较未转染组、空质粒转染组及pCMV-Myc-NGB质粒转染组的乳酸脱氢酶(LDH)活性及MTT细胞活性,证明了高表达NGB对机械性损伤后的神经源细胞具有保护作用。
4、大鼠急性局灶性脑损伤分级模型的建立:改进Feeney s自由落体模型装置并制作大鼠急性局灶性脑损伤模型,以不同冲击力(200gcm、600gcm、1000gcm)致大鼠脑损伤,鼠脑切片后并用TTC染色,采用Image J软件计算各组损伤后坏死脑组织的体积,经比较存在组间差异
5、大鼠急性局灶性颅脑损伤后血清NGB的水平变化:自行制备NGB双抗夹心ELISA检测试剂盒;测定伤后轻、中、重各组脑损伤血清中的NGB含量并比较差异,证明了血清中NGB水平脑损伤程度呈正相关;测定同一损伤程度下,损伤后各时间点血清NGB的水平,显示了损伤后血清中NGB水平的变化趋势。
综上所述,通过体外实验,本研究证实单纯的机械性损伤可以刺激神经元脑红蛋白表达上调,在机械性神经损伤中,脑红蛋白具有神经细胞保护功能,为基于此保护机制研发新的神经保护药物用于脑损伤的治疗提供了依据;通过在体实验,初步证明脑损伤后血清NGB水平与损伤程度的关系,及脑损伤后血清NGB水平的时间变化趋势,为临床上能够量化判断脑损伤的严重程度、转归与预后奠定了基础。
Oxygen is the essential element of life.The metabolism and utilization of oxygen are two important propositions in life science.It is well known that brain is one of major oxygen-consurning organ and sensitive to hypoxia.But if there exists a special globin for oxgen's metalolism and transport in brain is not known. Till 2000,Burmester reported a newly identified protein-neuroglobin(NGB), mainly expressed in nervous systems and retina of vertebrate,which is a monomer with high oxygen affinit5.Studies revealed NGB binded oxygen reversibly and played an important role in oxygen homeostasis of neural tissues. Other studies demonstrated that expression of NGB could be up-regulated under hypoxic conditions and had neuro-protective function both in vitro and in vivo. The discovery of NGB and the results of recent studies provide new opportunities to further understanding of neuroprotection and may lead to a novel therapeutic and diagnostic and therapeutic strategy for cerebral stroke and other neurodegenerated diseases.
However.relatively little is known about the expression changes and effect of neuroglobin in mechanical injury of nerve.We designed experiments in vitro and in vivo to research the subject as follows:
1、We cultured primary neuron in vitro after obtaining cerebral cortex from fetus rats brain and purifying it.Then the cultured neurons were injured mechanically through the model designed by ourselves,and the result of injury was proved by observing the changes under light microscope and dyeing immunohistochemically.
2、We choosed two methods to observe the expression changes of neurons NGB at different time points after mechanical injury:One was immunohistochemical method with image analysis technology using NIH software-Image J,the other was Western Blot technology.The two both demonstrated that the expression of NGB was up-regulated afer mechanical injury and reached the peak after 16 hours since injury.
3、Because of the low transfection efficiency of neuron,we choosed the neuroblastoma cell-B104 cell to undertake the experiments.Firstly,the B104 cell was proved to appear similar time course of NGB's expression change after mechanical injury using Western Blot technology.Then we used the pCMV-Myc-NGB plasmid to transfect the B104 cell and proved that the expression of NGB was more than the cell untrasfected or transfected by pure plasmid(pCMV-Myc plasmid).The viability of B104 cell transfected by pCMV-Myc-NGB plasmid was better than that of the cell untransfected or transtfected by pure plasmid through measuring LDH activity or MTT absorbance.
4、We set up acute local traumatic brain injury(TBI)graded model in rats using improved device of Feeney's weight-dropping model.The three groups of brain trauma were made by different impact(200g×cm、600g×cm、1000g×cm). Then the injured brain was cut sheets and dyeing with TTC.The volume of necrotic brain tissue were calculated by NIH software-Image J and proved to be different statistically among different-impact groups.
5、We measured the NGB's levels in the rat serum at the same time point of the mild,moderate and severe TBI using the home-made ELISA kit.The serum levels of NGB were different statistically among different grade TBI.Also we measured the serum NGB's levels at different time points after TBI of the same grade to explore the time-course of NGB's serum concentration.
In summary,the NGB's expression was up-regulated by the mechanical injury in neuron and NGB had neuroprotection effection.And NGB's serum level was relative to the severity of TBI.it may be a new diagnostic tool to judge the TBI's severity.
在神经外科的临床工作中,颅脑创伤是最常见的病种之一。创伤后脑组织水肿,对氧的摄取和利用明显下降是引起脑损伤的重要因素。而NGB作为神经系统特有的携氧球蛋白,研究其在神经系统机械性损伤中的表达和作用,将为临床工作中对颅脑创伤的诊断、治疗提供新的契机和切入点。本研究包括:
1、建立体外培养原代神经元机械性损伤模型:手术取胎鼠人脑皮层,分离纯化后作原代神经元培养:用抗神元特异性蛋白——神经丝蛋白(Neurofilament protein,NF)抗体鉴定细胞纯度;对Banker体外损伤模型进行改良,采用自行设计的模版对培养神经元进行机械性损伤,并在损伤后行光镜下观察及免疫组化检测c-Fos蛋白来证实神经元的损伤。
2、分别采用免疫组织化学、Western Blot技术检测神经元损伤后NGB的表达变化:神经元NGB染色采用链霉素抗生物素-过氧化物酶连接法(SP法),并采用图像分析技术(Image J软件,美国国立卫生院--NIH)观测损伤前、后不同时间点神经元的NGB免疫组化反应阳性信号的灰度值;细胞裂解提取蛋白后,采用Western Blot技术检测神经元损伤前、后各时间点NGB的表达。两种检测方法的结果一致。
3、增加NGB的表达对损伤神经源性细胞活性的影响:因神经元的转染率低,因而采用转染率高的神经源性细胞(B104细胞)进行实验。先按照损伤模型对B104细胞进行机械性损伤,采用Western Blot技术检测B104细胞损伤后NGB的表达变化,确定其变化趋势与原代神经元相同;然后采用军事医学科学院二所十三室制备的pCMV-Myc-NGB质粒转染B104细胞,用Western Blot检测技术验证B104细胞转染pCMV-Myc-NGB质粒后NGB表达增多:选取损伤后0.5h、2h两个时间点,分别测定并比较未转染组、空质粒转染组及pCMV-Myc-NGB质粒转染组的乳酸脱氢酶(LDH)活性及MTT细胞活性,证明了高表达NGB对机械性损伤后的神经源细胞具有保护作用。
4、大鼠急性局灶性脑损伤分级模型的建立:改进Feeney s自由落体模型装置并制作大鼠急性局灶性脑损伤模型,以不同冲击力(200gcm、600gcm、1000gcm)致大鼠脑损伤,鼠脑切片后并用TTC染色,采用Image J软件计算各组损伤后坏死脑组织的体积,经比较存在组间差异
5、大鼠急性局灶性颅脑损伤后血清NGB的水平变化:自行制备NGB双抗夹心ELISA检测试剂盒;测定伤后轻、中、重各组脑损伤血清中的NGB含量并比较差异,证明了血清中NGB水平脑损伤程度呈正相关;测定同一损伤程度下,损伤后各时间点血清NGB的水平,显示了损伤后血清中NGB水平的变化趋势。
综上所述,通过体外实验,本研究证实单纯的机械性损伤可以刺激神经元脑红蛋白表达上调,在机械性神经损伤中,脑红蛋白具有神经细胞保护功能,为基于此保护机制研发新的神经保护药物用于脑损伤的治疗提供了依据;通过在体实验,初步证明脑损伤后血清NGB水平与损伤程度的关系,及脑损伤后血清NGB水平的时间变化趋势,为临床上能够量化判断脑损伤的严重程度、转归与预后奠定了基础。
Oxygen is the essential element of life.The metabolism and utilization of oxygen are two important propositions in life science.It is well known that brain is one of major oxygen-consurning organ and sensitive to hypoxia.But if there exists a special globin for oxgen's metalolism and transport in brain is not known. Till 2000,Burmester reported a newly identified protein-neuroglobin(NGB), mainly expressed in nervous systems and retina of vertebrate,which is a monomer with high oxygen affinit5.Studies revealed NGB binded oxygen reversibly and played an important role in oxygen homeostasis of neural tissues. Other studies demonstrated that expression of NGB could be up-regulated under hypoxic conditions and had neuro-protective function both in vitro and in vivo. The discovery of NGB and the results of recent studies provide new opportunities to further understanding of neuroprotection and may lead to a novel therapeutic and diagnostic and therapeutic strategy for cerebral stroke and other neurodegenerated diseases.
However.relatively little is known about the expression changes and effect of neuroglobin in mechanical injury of nerve.We designed experiments in vitro and in vivo to research the subject as follows:
1、We cultured primary neuron in vitro after obtaining cerebral cortex from fetus rats brain and purifying it.Then the cultured neurons were injured mechanically through the model designed by ourselves,and the result of injury was proved by observing the changes under light microscope and dyeing immunohistochemically.
2、We choosed two methods to observe the expression changes of neurons NGB at different time points after mechanical injury:One was immunohistochemical method with image analysis technology using NIH software-Image J,the other was Western Blot technology.The two both demonstrated that the expression of NGB was up-regulated afer mechanical injury and reached the peak after 16 hours since injury.
3、Because of the low transfection efficiency of neuron,we choosed the neuroblastoma cell-B104 cell to undertake the experiments.Firstly,the B104 cell was proved to appear similar time course of NGB's expression change after mechanical injury using Western Blot technology.Then we used the pCMV-Myc-NGB plasmid to transfect the B104 cell and proved that the expression of NGB was more than the cell untrasfected or transfected by pure plasmid(pCMV-Myc plasmid).The viability of B104 cell transfected by pCMV-Myc-NGB plasmid was better than that of the cell untransfected or transtfected by pure plasmid through measuring LDH activity or MTT absorbance.
4、We set up acute local traumatic brain injury(TBI)graded model in rats using improved device of Feeney's weight-dropping model.The three groups of brain trauma were made by different impact(200g×cm、600g×cm、1000g×cm). Then the injured brain was cut sheets and dyeing with TTC.The volume of necrotic brain tissue were calculated by NIH software-Image J and proved to be different statistically among different-impact groups.
5、We measured the NGB's levels in the rat serum at the same time point of the mild,moderate and severe TBI using the home-made ELISA kit.The serum levels of NGB were different statistically among different grade TBI.Also we measured the serum NGB's levels at different time points after TBI of the same grade to explore the time-course of NGB's serum concentration.
In summary,the NGB's expression was up-regulated by the mechanical injury in neuron and NGB had neuroprotection effection.And NGB's serum level was relative to the severity of TBI.it may be a new diagnostic tool to judge the TBI's severity.
引文
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22 Lathouder Y, Sakaguchi D S, Ostojic J, et al.Neuroglobin and cytoglobin: oxygen-binding proteins in retinal neurons. Invest Ophthalmol Vis Sci, 2006 Mar.47(3): 1016-23.
23 Qiu Y. Sutton L. Riggs A F. et al.Identification of myoglobin in human smooth muscle. J Biol Chem. 1998, 273:23426-23432
24 Reuss S. Schmidt M, Bentmann A. et al.Divergent distribution in vascular and avascular mammalian retinae links neuroglobin to cellular respiration. J Biol Chem. 2005 May.280(21):20660-5.
25 Brunori, M. Nitric oxide moves myoglobin centre stage. Trends Biochem Sci,2001.26:209-210.
26 Fago A, Mathevvs AJ, Moens L, et al. The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c. FEBS Lett, 2006 Sep .580(20):4884-8.
27 Flogel U, Merx M W, Godecke A, et al. Myoglobin: A scavenger of bioactive NO. Proc Natl Acad Sci USA,2001,98 (9) :735-740
28 Nienhaus K, Giuffre A, Brunori M. et al. Neuroglobin, nitric oxide, and oxygen: functional pathways and conformational changes. Proc Natl Acad Sci USA,2005 Jun,102(24):8483-8.
29 徐文琳,王春丽,张永亮等。脑红蛋白与Na~+-K~+ATP酶β_2亚基的相互作用及作用位点的鉴定.生物化学及生物物理学报.2003,35(9):823-828
30 Wakasugi K,Nakano T,Morishima I.Association of human neuroglobin with cystatin.a cysteine proteinase inhibitor.Biochemistry,2004,43:5119-5125.
31 Schwindinger WF.Robishaw JD.Heterotrimeric G-protein βγ-dimers in growth and differentiation.Oncogene,2001.20:1653-1660.
32 Wakasugi K,Nakano T,Morishima I,et al.Oxidized human neuroglobin as a heterotrimeric GR protein guanine nucleotide dissociation inhibitor.J Biol Chem.2003.278:36505-36512.
33 Wakasugi K.Morishima I.Identification of residues in human neuroglobin crucial for Guanine nucleotide dissociation inhibitor activity.Biochemistry,2005 Mar,44(8):2943-8.
34 Kim H E,Du F.Fang M.et al.Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-l.Proc Natl Acad Sci USA,2005.102:17545-17550.
35 Suto D,Sato K.Ohba Y.et al.Suppression of the pro-apoptotic function of cytochrome c by singlet oxygen via a haem redox state-independent mechanism.Biochem J,2005.392:399-406.
36 Fuchs C,Heib V,Kiger L,et al.Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure,expression pattern,and ligand binding.J Biol Chem,2004,279:24116-24122.
1 Buchan A, Pulsinelli WA. Hypothermia but not the N-methyl-D-aspartate antagonist,MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia. JNeurosci. 1999 Jan; 10(1):311-6.
2 Hovda DA, Becker DP, Katayama Y. Secondary injury and acidosis J Neurotrauma. 2000 Mar;9 Suppl 1 :S47-60
3 Tecoma ES, Monyer H, Goldberg MP, Choi DW. Traumatic neuronal injury in vitro is attenuated by NMDA antagonists. Neuron. 2005 Jun:2(6): 1541-5
4 Mukhin AG. Ivanova SA, Knoblach SM, Faden Al. New in vitro model of traumatic neuronal injury: evaluation of secondary injury and glutamate receptor-mediated neurotoxicity. J Neurotrauma. 2007 Sep:14(9):651-63
5 Gross GW, Lucas JH. Higgins ML. Laser microbeam surgery: ultrastructural changes associated with neurite transection in culture. J Neurosci. 2003 Oct;3( 10): 1979-93
6 Balentine JD. Greene WB, Bomstein M. In vitro spinal cord trauma. Lab Invest. 1999 Jan:58(1):93-9
7 Murphy EJ. Horrocks LA. A model for compression trauma: pressure-induced injury in cell cultures. J Neurotrauma. 1993 Winter; 10(4):431-44
8 Margulies SS, Thibault LE, Gennarelli TA. Physical model simulations of brain injury in the primate. J Biomech. 1990;23(8):823-36
9 LaPlaca MC, Thibault LE. An in vitro traumatic injury model to examine the response of neurons to a hydrodynamically-induced deformation. Ann Biomed Eng. 1997 Jul-Aug;25(4):665-77
10 Ellis EF, McKinney JS, Willoughby KA. A new model for rapid stretch-induced injury of cells in culture: characterization of the model using astrocytes. J Neurotrauma. 1995 Jun;12(3):325-39
11 Morrison B 3rd, Meaney DF, McIntosh TK. Mechanical characterization of an in vitro device designed to quantitatively injure living brain tissue. Ann Biomed Eng. 1998 May-Jun;26(3):381 -90
12 Kirkpatrick JB, Higgins ML, Lucas JH. In vitro simulation of neural trauma by laser. J Neuropathoi Exp Neurol. 1999 May;44(3):268-84
13 Sun Y, Jin K,Mao X O, et al.Neuroglobin protects the brain from experimental stroke in vivo. Proc Natl Acad Sci USA, 2003,100: 3497-3500.
14 Charles AC. Glia-neuron intercellular calcium signaling. Dev Neurosci. 1999;16(3-4): 196-206
15 Wallis RA. Panizzon KL. Felbamate neuroprotection against CA1 traumatic neuronal injury. Eur J Pharmacol. 1995 Dec 29;294(2-3):475-82
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17 Rosenberg LJ, Lucas JH. Reduction of NaCl increases survival of mammalian spinal neurons subjected to dendrite transection injury. Brain Res. 1996 Sep 23:734(1-2):349-53
18 Lefrancois T, Fages C, Peschanski M. Neuritic outgrowth associated with astroglial phenotypic changes induced by antisense glial fibrillary acidic protein (GFAP) mRNA in injured neuron-astrocyte cocultures. J Neurosci. 2007 Jun 1;17(11):4121-8
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1 Burmester T,Weich B,Reinhardt S,et al.A vertebrate globin expressed in the brain.Nature.2000,407(6803):520-523.
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13 Sun Y,Jin K,Mao X O.et al.Neuroglobin protects the brain from experimental stroke in vivo.Proc Natl Acad Sci USA,2003,100:3497-3500.
14 Hardison R C.A brief history of hemoglobins:plant,animal,protist,and bacteria.Proc Natl Acad Sci USA,1996,93:5675-5679.
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18 Dickerson R E ,Geis I. Hemoglobin: Structure, function, evolution, and pathology (Benjamin/Cummings, Menlo Park. California, 1983):757-759.
19 Stryer L. Biochemistry (Freeman. New York. 1995):931 -936.
20 Couture M.Burmester T.Hankeln T,et al.The heme environment of mouse neuroglobin.Evidence for the presence of two conformations of the heme pocket.J Biol Chem,2001.276(39):36377-82.
21 Garry D J. Ordway G A. Lorenz J N. et al.Mice without myoglobin. Nature. 1998. 395:905-908.
22 Lathouder Y, Sakaguchi D S, Ostojic J, et al.Neuroglobin and cytoglobin: oxygen-binding proteins in retinal neurons. Invest Ophthalmol Vis Sci, 2006 Mar.47(3): 1016-23.
23 Qiu Y. Sutton L. Riggs A F. et al.Identification of myoglobin in human smooth muscle. J Biol Chem. 1998, 273:23426-23432
24 Reuss S. Schmidt M, Bentmann A. et al.Divergent distribution in vascular and avascular mammalian retinae links neuroglobin to cellular respiration. J Biol Chem. 2005 May.280(21):20660-5.
25 Brunori, M. Nitric oxide moves myoglobin centre stage. Trends Biochem Sci,2001.26:209-210.
26 Fago A, Mathevvs AJ, Moens L, et al. The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c. FEBS Lett, 2006 Sep .580(20):4884-8.
27 Flogel U, Merx M W, Godecke A, et al. Myoglobin: A scavenger of bioactive NO. Proc Natl Acad Sci USA,2001,98 (9) :735-740
28 Nienhaus K, Giuffre A, Brunori M. et al. Neuroglobin, nitric oxide, and oxygen: functional pathways and conformational changes. Proc Natl Acad Sci USA,2005 Jun,102(24):8483-8.
29 徐文琳,王春丽,张永亮等。脑红蛋白与Na~+-K~+ATP酶β_2亚基的相互作用及作用位点的鉴定.生物化学及生物物理学报.2003,35(9):823-828
30 Wakasugi K,Nakano T,Morishima I.Association of human neuroglobin with cystatin.a cysteine proteinase inhibitor.Biochemistry,2004,43:5119-5125.
31 Schwindinger WF.Robishaw JD.Heterotrimeric G-protein βγ-dimers in growth and differentiation.Oncogene,2001.20:1653-1660.
32 Wakasugi K,Nakano T,Morishima I,et al.Oxidized human neuroglobin as a heterotrimeric GR protein guanine nucleotide dissociation inhibitor.J Biol Chem.2003.278:36505-36512.
33 Wakasugi K.Morishima I.Identification of residues in human neuroglobin crucial for Guanine nucleotide dissociation inhibitor activity.Biochemistry,2005 Mar,44(8):2943-8.
34 Kim H E,Du F.Fang M.et al.Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-l.Proc Natl Acad Sci USA,2005.102:17545-17550.
35 Suto D,Sato K.Ohba Y.et al.Suppression of the pro-apoptotic function of cytochrome c by singlet oxygen via a haem redox state-independent mechanism.Biochem J,2005.392:399-406.
36 Fuchs C,Heib V,Kiger L,et al.Zebrafish reveals different and conserved features of vertebrate neuroglobin gene structure,expression pattern,and ligand binding.J Biol Chem,2004,279:24116-24122.
1 Buchan A, Pulsinelli WA. Hypothermia but not the N-methyl-D-aspartate antagonist,MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia. JNeurosci. 1999 Jan; 10(1):311-6.
2 Hovda DA, Becker DP, Katayama Y. Secondary injury and acidosis J Neurotrauma. 2000 Mar;9 Suppl 1 :S47-60
3 Tecoma ES, Monyer H, Goldberg MP, Choi DW. Traumatic neuronal injury in vitro is attenuated by NMDA antagonists. Neuron. 2005 Jun:2(6): 1541-5
4 Mukhin AG. Ivanova SA, Knoblach SM, Faden Al. New in vitro model of traumatic neuronal injury: evaluation of secondary injury and glutamate receptor-mediated neurotoxicity. J Neurotrauma. 2007 Sep:14(9):651-63
5 Gross GW, Lucas JH. Higgins ML. Laser microbeam surgery: ultrastructural changes associated with neurite transection in culture. J Neurosci. 2003 Oct;3( 10): 1979-93
6 Balentine JD. Greene WB, Bomstein M. In vitro spinal cord trauma. Lab Invest. 1999 Jan:58(1):93-9
7 Murphy EJ. Horrocks LA. A model for compression trauma: pressure-induced injury in cell cultures. J Neurotrauma. 1993 Winter; 10(4):431-44
8 Margulies SS, Thibault LE, Gennarelli TA. Physical model simulations of brain injury in the primate. J Biomech. 1990;23(8):823-36
9 LaPlaca MC, Thibault LE. An in vitro traumatic injury model to examine the response of neurons to a hydrodynamically-induced deformation. Ann Biomed Eng. 1997 Jul-Aug;25(4):665-77
10 Ellis EF, McKinney JS, Willoughby KA. A new model for rapid stretch-induced injury of cells in culture: characterization of the model using astrocytes. J Neurotrauma. 1995 Jun;12(3):325-39
11 Morrison B 3rd, Meaney DF, McIntosh TK. Mechanical characterization of an in vitro device designed to quantitatively injure living brain tissue. Ann Biomed Eng. 1998 May-Jun;26(3):381 -90
12 Kirkpatrick JB, Higgins ML, Lucas JH. In vitro simulation of neural trauma by laser. J Neuropathoi Exp Neurol. 1999 May;44(3):268-84
13 Sun Y, Jin K,Mao X O, et al.Neuroglobin protects the brain from experimental stroke in vivo. Proc Natl Acad Sci USA, 2003,100: 3497-3500.
14 Charles AC. Glia-neuron intercellular calcium signaling. Dev Neurosci. 1999;16(3-4): 196-206
15 Wallis RA. Panizzon KL. Felbamate neuroprotection against CA1 traumatic neuronal injury. Eur J Pharmacol. 1995 Dec 29;294(2-3):475-82
16 Lucas JH, Emery DG, Wang G. In vitro investigations of the effects of nonfreezing low temperatures on lesioned and uninjured mammalian spinal neurons. J Neurotrauma. 1994 Feb; 11(1 ):35-61
17 Rosenberg LJ, Lucas JH. Reduction of NaCl increases survival of mammalian spinal neurons subjected to dendrite transection injury. Brain Res. 1996 Sep 23:734(1-2):349-53
18 Lefrancois T, Fages C, Peschanski M. Neuritic outgrowth associated with astroglial phenotypic changes induced by antisense glial fibrillary acidic protein (GFAP) mRNA in injured neuron-astrocyte cocultures. J Neurosci. 2007 Jun 1;17(11):4121-8