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NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons
原代培养神经细胞中的NMDA诱导兴奋毒性和乳酸脱氢酶试验   

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Abstract

N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitotoxicity is thought to contribute to the pathogenesis of a large number of chronic neurodegenerative disorders (such as Alzheimer’s and Huntington’s diseases to mental illnesses) in addition to acute brain insults such as stroke and brain trauma. Understanding the mechanisms underlying NMDAR-mediated excitotoxicity may lead to development of novel therapeutics for treating neurological diseases. Stimulation of primary cultured neurons with excessive NMDA is widely used as an in vitro model for studying NMDAR-mediated excitotoxicity, which allows careful dissection of the detailed cellular mechanisms underlying excitotoxic neuronal death.
Lactate dehydrogenase (LDH) is a cytoplasmic enzyme which can convert NAD into NADH. LDH is released from cells into culture medium when the plasma membrane integrity is compromised. Therefore, the amount of released LDH represents the degree of cell death. In our current study, the extracellular LDH level was measured using an in vitro Toxicology Assay Kit obtained from Sigma-Aldrich. The basis of this LDH assay is: 1) LDH reduces NAD into NADH, 2) the resulting NADH is then utilized in the stoichiometric conversion of a tetrazolium dye, and 3) the resulting colored compound is measured by a spectrophotometric microplate reader at a wavelength of 490 nm. The cell death rate was expressed as a percentage (%) between the absorbance of treated group and that of control group.

Keywords: NMDAR(NMDA受体), Excitotoxicity(兴奋性毒性), Neuronal cultures(神经细胞), LDH assay(LDH法), Rat(老鼠)

Materials and Reagents

  1. Primary cultured neurons
  2. NMDA (Sigma-Aldrich, catalog number: M3262-25MG )
  3. Neurobasal medium (Life Technologies, Invitrogen™, catalog number: 21103 )
  4. LDH Assay Substrate Solution
  5. LDH Assay Dye Solution
  6. LDH Assay Cofactor Preparation
  7. In vitro Toxicology Assay Kit (Sigma-Aldrich, catalog number: TOX-7 )

Equipment

  1. 37 °C, 5% CO2 Cell culture incubator
  2. 96-well plate (Sigma-Aldrich, catalog number: SIAL0596-50EA )
  3. Spectrometer for 96 well plate that can measure 490 nm and 690 nm (Molecular Devices, model:  SpectraMax M2e Multi-Mode Microplate Reader)

Procedure

  1. NMDA-induced Excitotoxicity
    1. Prepare fresh NMDA 1,000x stock solution (25 mM) with fresh neurobasal medium. Old NMDA stock solution is less effective in inducing excitotoxicity, and higher dose might be required.
    2. Immediately prior to NMDA treatment, half of the conditioned medium (old medium) is taken out and placed in the incubator to keep warm and pH balanced. The conditional medium taken out is saved for replacement of NMDA-containing medium after the excitotoxicity treatment. The reason to use conditional medium rather than fresh medium for replacement is that primary culture neurons are very sensitive to environment change, and using the conditional medium can minimize additional stimulation to neurons other than the NMDA treatment during the whole procedure.
    3. NMDA is added directly to the culture medium to initiate excitotoxicity stimulation of neurons. The working concentration of NMDA is 25 μM. If the culture medium in the plate is 10 ml, the NMDA stock (25 mM) added will be 1/1,000 of 10 ml, which is 10 μl. Neurons are kept in the incubator during the treatment.
    4. After 60 min incubation with NMDA, neurons are washed with fresh neural basal medium (warm and pH balanced) for once and then returned to the previously saved conditional medium.
    5. Neurons are allowed to recover for different periods of time, ranging from 0 h to 24 h until further experiments.
    6. Significant neuronal death could be observed after 6 h recovery and reaches its maximum level after 24 h.  
      Note: The conditioned medium is essential to minimize the stress for neurons.

  2. Lactate dehydrogenase assay to detect cell death
    1. Prepare the Lactate Dehydrogenase Assay Mixture immediately before performing the assay by mixing equal volume of LDH Assay Substrate Solution, LDH Assay Dye Solution and LDH Assay Cofactor Preparation according to Sigma manufacture protocol. Store the mixture on ice.
    2. Remove cultures from incubator.
    3. Transfer proximately 100 μl culture medium from each condition into a micro-centrifuge tube respectively and spin down the culture medium at 13,000 rpm for 1 min to deposit the cell debris.
    4. Transfer 50 μl cultured medium from each condition into 96 well plate. Add 100 μl Lactate Dehydrogenase Assay Mixture to each sample. The volume of culture medium and Lactate Dehydrogenase Assay Mixture could be adjusted proportionally.
    5. Cover the plate with Aluminum Foil to avoid light exposure.
    6. Incubate at room temperature for several minutes to several hours depending on the concentration of LDH in the culture medium. For the first time user, reading the results every 20 min to determine the optimal end point is highly recommended. To obtain comparable results between different batches of cultures, a similar end point should be used. As the culture conditions (cell viability, cell density and medium volume) vary across different batches of neurons, it is sometimes hard to determine the end point using the length of reaction time. For example, in one batch of neurons, it may take 1 h to achieve a reading of 1.0 at 490 nm in the control group (non-treated group); while in another batch, it takes 1.5 h. Therefore, it’s highly recommended that the reaction should be read every 20 min during the whole LDH reaction, and only the data collected at the point when the reading of control group (e.g. 1.0 ± 0.1) is comparable to other batches should be used. As the reading is just arbitrary number, the data is only meaningful when comparing the treatment group with its control group.
    7. Spectrophotometrically measure absorbance at a wavelength of 490 nm. Measure the background absorbance of 96 well plate at 690 nm. Subtract reading of 690 nm from that of 490 nm to obtain the final reading.
    8. If plate reader is not available, samples could be transferred to appropriate sized cuvettes for spectrophotometric measurement.

Notes

  1. The concentration of extracellular LDH depends on cell viability, cell density and medium volume. Ideally, cells density and medium volume in different culture wells should be exactly the same to avoid fluctuation of the results.
  2. Some culture medium contains a significant level of LDH activity. In this case, blank medium should be measured and subtracted from the final results. Alternatively, serum-containing medium could be replaced with serum-free medium before any treatment.

Acknowledgments

This work was supported by the Canadian Institutes of Health Research (CIHR), CHDI Foundation, the Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111– 004), the National Research Council of Taiwan (NSC100-2632-B-039-001-MY3 and NSC 101-2320-B-039-059-), the National Natural Science Foundation of China 31040085 and 81271221, and Chongqing International Science and Technology Cooperation Foundation cstc201110003. Y.T.W. is a Howard Hughes Medical Institute International Scholar, and Heart and Stroke Foundation of British Columbia and Yukon Chair in Stroke Research. We thank Yuping Li, Dr. Henry Martin, Dr. Lidong Liu, and Dr. Jie Lu for technical support.

References

  1. Decker, T. and Lohmann-Matthes, M. L. (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods 115(1): 61-69.   
  2. Legrand, C., Bour, J. M., Jacob, C., Capiaumont, J., Martial, A., Marc, A., Wudtke, M., Kretzmer, G., Demangel, C., Duval, D. and et al. (1992). Lactate dehydrogenase (LDH) activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium [corrected]. J Biotechnol 25(3): 231-243. 
  3. Liu, Y., Wong, T. P., Aarts, M., Rooyakkers, A., Liu, L., Lai, T. W., Wu, D. C., Lu, J., Tymianski, M., Craig, A. M. and Wang, Y. T. (2007). NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J Neurosci 27(11): 2846-2857.
  4. Taghibiglou, C., Martin, H. G., Lai, T. W., Cho, T., Prasad, S., Kojic, L., Lu, J., Liu, Y., Lo, E., Zhang, S., Wu, J. Z., Li, Y. P., Wen, Y. H., Imm, J. H., Cynader, M. S. and Wang, Y. T. (2009). Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries. Nat Med 15(12): 1399-1406.
  5. Zhang, S., Taghibiglou, C., Girling, K., Dong, Z., Lin, S. Z., Lee, W., Shyu, W. C. and Wang, Y. T. (2013). Critical role of increased PTEN nuclear translocation in excitotoxic and ischemic neuronal injuries. J Neurosci 33(18): 7997-8008.    
  6. Taghibiglou, C., Martin, H. G., Lai, T. W., Cho, T., Prasad, S., Kojic, L., Lu, J., Liu, Y., Lo, E., Zhang, S., Wu, J. Z., Li, Y. P., Wen, Y. H., Imm, J. H., Cynader, M. S. and Wang, Y. T. (2009). Role of NMDA receptor-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries. Nat Med 15(12): 1399-1406.

简介

认为N-甲基-D-天门冬氨酸受体(NMDAR)介导的兴奋性毒性被认为有助于大量慢性神经退行性疾病(如阿尔茨海默病和亨廷顿疾病对精神疾病)的发病机制,除了急性脑损伤如中风和脑外伤。了解NMDAR介导的兴奋性毒性的机制可能导致治疗神经系统疾病的新疗法的发展。具有过量NMDA的原代培养神经元的刺激被广泛用作研究NMDAR介导的兴奋性毒性的体外模型,其允许仔细解剖兴奋性毒性神经元死亡的详细细胞机制。
乳酸脱氢酶(LDH)是可将NAD转化为NADH的细胞质酶。当质膜完整性受损时,LDH从细胞释放到培养基中。因此,释放的LDH的量代表细胞死亡的程度。在我们目前的研究中,使用从Sigma-Aldrich获得的体外毒理学测定试剂盒测量细胞外LDH水平。该LDH测定的基础是:1)LDH将NAD还原成NADH,2)然后将所得NADH用于四唑鎓染料的化学计量转化,和3)所得着色化合物通过分光光度计酶标仪在波长为490nm。细胞死亡率以处理组的吸光度与对照组的吸光度之间的百分比(%)表示。

关键字:NMDA受体, 兴奋性毒性, 神经细胞, LDH法, 老鼠

材料和试剂

  1. 原代培养神经元
  2. NMDA(Sigma-Aldrich,目录号:M3262-25MG)
  3. Neurobasal培养基(Life Technologies,Invitrogen TM,目录号:21103)
  4. LDH测定底物溶液
  5. LDH测定染料溶液
  6. LDH测定辅因子准备
  7. 体外毒理学测定试剂盒(Sigma-Aldrich,目录号:TOX-7)

设备

  1. 37℃,5%CO 2细胞培养箱
  2. 96孔板(Sigma-Aldrich,目录号:SIAL0596-50EA)
  3. 可测量490nm和690nm的96孔板的分光计(Molecular Devices,型号:SpectraMax M2e多模式微板读数器)

程序

  1. NMDA诱导的兴奋毒性
    1. 用新鲜的神经基础培养基制备新鲜NMDA 1,000x储备溶液(25mM)。旧NMDA储备溶液在诱导兴奋性毒性方面不太有效,并且可能需要更高的剂量。
    2. 在NMDA处理之前,取出一半条件培养基(旧培养基),并置于培养箱中以保持温热和pH平衡。在兴奋性毒性处理后,取出的条件培养基被保存以替换含NMDA的培养基。使用条件培养基而不是新鲜培养基进行替换的原因是原代培养神经元对环境变化非常敏感,并且使用条件培养基可以在整个程序期间使对除NMDA处理之外的神经元的额外刺激最小化。
    3. 将NMDA直接加入到培养基中以启动神经元的兴奋性毒性刺激。 NMDA的工作浓度为25μM。如果平板中的培养基为10ml,则将NMDA原液(25μl) mM)将为10μl的1/1000,其为10μl。 治疗期间神经元保持在培养箱中。
    4. 与NMDA孵育60分钟后,用新鲜的神经基础培养基(温和和pH平衡)洗涤神经元一次,然后返回到先前保存的条件培养基中。
    5. 允许神经元恢复不同的时间段,范围从0小时至24小时,直到进一步的实验
    6. 在6小时恢复后可观察到显着的神经元死亡,并在24小时后达到其最大水平。  
      注意:条件培养基对于使神经元的应激最小化是必需的。

  2. 乳酸脱氢酶测定法检测细胞死亡
    1. 在进行测定之前立即通过混合等体积的LDH测定底物溶液,LDH测定染料溶液和LDH测定辅因子制备根据Sigma制造方案制备乳酸脱氢酶测定混合物。将混合物储存在冰上。
    2. 从培养箱中取出培养物。
    3. 将每种条件下的大约100μl培养基分别转移到微离心管中,并以13,000rpm离心培养基1分钟以沉积细胞碎片。
    4. 转移50μl培养基从每个条件到96孔板。向每个样品中加入100μl乳酸脱氢酶测定混合物。可以按比例调节培养基和乳酸脱氢酶测定混合物的体积。
    5. 用铝箔覆盖板以避免曝光。
    6. 根据培养基中LDH的浓度,在室温下孵育几分钟至几小时。对于第一次使用者,每20分钟读取结果以确定最佳终点是强烈推荐。为了在不同批次的培养物之间获得相当的结果,应当使用类似的终点。随着培养条件(细胞活力,细胞密度和培养基体积)的变化 不同批次的神经元,有时难以使用反应时间的长度确定终点。例如,在一批神经元中,在对照组(未处理组)中在490nm处获得1.0的读数可能需要1小时;而在另一批次中,需要1.5小时。因此,强烈推荐在整个LDH反应期间每20分钟读取反应,并且只有当对照组的读数(例如,1.0±0.1)与对照组的读数相当时才收集的数据其他批次应使用。由于读数只是任意数,所以数据仅在将治疗组与其对照组比较时才有意义。
    7. 分光光度法测量在490nm波长处的吸光度。测量96孔板在690nm的背景吸光度。从490nm处减去690nm的读数,以获得最终读数
    8. 如果没有读板器,样品可以转移到适当尺寸的比色皿用于分光光度测量

笔记

  1. 细胞外LDH的浓度取决于细胞活力,细胞密度和培养基体积。理想情况下,不同培养孔中的细胞密度和培养基体积应完全相同,以避免结果的波动
  2. 一些培养基含有显着水平的LDH活性。在这种情况下,应测量空白培养基并从最终结果中减去。或者,在任何治疗之前,可以用无血清培养基替换含血清培养基。

致谢

这项工作由加拿大卫生研究院(CIHR),CHDI基金会,台湾卫生临床试验和研究中心(DOH102-TD-B-111- 004),台湾国家研究委员会(NSC100 -2632-B-039-001-MY3和NSC 101-2320-B-039-059-),中国国家自然科学基金31040085和81271221,以及重庆国际科技合作基金会cstc201110003。 Y.T.W.是霍华德休斯医学研究所国际学者,不列颠哥伦比亚省心脏和卒中基金会以及中风研究中的育空座椅。我们感谢李玉平,Henry Martin博士,刘丽东博士和陆杰博士的技术支持。

参考文献

  1. Decker,T。和Lohmann-Matthes,M.L。(1988)。 在测量细胞毒性和肿瘤坏死因子中定量乳酸脱氢酶释放的快速而简单的方法(TNF)活性。 Immunol Methods 115(1):61-69。   
  2. Legrand,C.,Bour,JM,Jacob,C.,Capiaumont,J.,Martial,A.,Marc,A.,Wudtke,M.,Kretzmer,G.,Demangel,C.,Duval, et al。 (1992)。 培养的真核细胞的乳酸脱氢酶(LDH)活性作为细胞中死细胞数量的标记中等[校正]。生物技术 25(3):231-243。 
  3. Liu,Y.,Wong,TP,Aarts,M.,Rooyakkers,A.,Liu,L.,Lai,TW,Wu,DC,Lu,J.,Tymianski,M.,Craig,AMand Wang,YT 2007)。 NMDA受体亚基在体外介导兴奋性毒性神经元死亡中具有不同的作用 27(11):2846-2857。
  4. Taghibiglou,C.,Martin,HG,Lai,TW,Cho,T.,Prasad,S.,Kojic,L.,Lu,J.,Liu,Y.,Lo,E.,Zhang, JZ,Li,YP,Wen,YH,Imm,JH,Cynader,MS和Wang,YT(2009)。 NMDA受体依赖性激活SREBP1在兴奋性毒性和缺血性神经元损伤中的作用。 em> Nat Med 15(12):1399-1406。
  5. Zhang,S.,Taghibiglou,C.,Girling,K.,Dong,Z.,Lin,S.Z.,Lee,W.,Shyu,W.C.and Wang,Y.T。 在兴奋性中毒和缺血性神经元损伤中增加PTEN核易位的关键作用。 J Neurosci 33(18):7997-8008。    
  6. Taghibiglou,C.,Martin,HG,Lai,TW,Cho,T.,Prasad,S.,Kojic,L.,Lu,J.,Liu,Y.,Lo,E.,Zhang, JZ,Li,YP,Wen,YH,Imm,JH,Cynader,MS和Wang,YT(2009)。 NMDA受体依赖性激活SREBP1在兴奋性毒性和缺血性神经元损伤中的作用。 em> Nat Med 15(12):1399-1406。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhang, S. and Wang, Y. T. (2013). NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons. Bio-protocol 3(21): e965. DOI: 10.21769/BioProtoc.965.
  2. Zhang, S., Taghibiglou, C., Girling, K., Dong, Z., Lin, S. Z., Lee, W., Shyu, W. C. and Wang, Y. T. (2013). Critical role of increased PTEN nuclear translocation in excitotoxic and ischemic neuronal injuries. J Neurosci 33(18): 7997-8008.    
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