Mukhin A G, Ivanova S A, Allen J W, Faden A I
Institute for Cognitive and Computational Sciences, Georgetown University Medical Center, Washington, DC 20007, USA.
J Neurosci Res. 1998 Mar 15;51(6):748-58. doi: 10.1002/(SICI)1097-4547(19980315)51:6<748::AID-JNR8>3.0.CO;2-B.
In vitro models of traumatic injury are useful adjuncts to animal models for studying mechanisms of post-traumatic cell death. Here we describe a new in vitro model in which reproducible levels of injury are delivered by a punch device that produces 28 parallel cuts in individual wells of 96-well microplates. Cell loss is measured by LDH assay or quantitative fluorometric assay for ethidium homodimer staining. Glial cultures show cell death restricted to the initial injury site, whereas neuronal/glial cultures demonstrate substantial spread of cell loss over time. We used this model to examine the role of pH and NMDA receptors in delayed post-traumatic injury. NMDA receptor blockade by dizocilpine (MK-801) or treatment with antisense oligodeoxynucleotides directed against NMDAR1 was neuroprotective. Decreased cell death was observed under acidic conditions whereas increased extracellular pH was associated with increased, MK-801 sensitive cell loss. Advantages of our model include: reproducible trauma induction; rapid measurements of cell injury; and use of 96-well microplates which reduce time and cost. This model appears to be well-suited for the study of selected mechanisms of post-traumatic neuronal injury as well as for screening potential neuroprotective agents.
创伤性损伤的体外模型是研究创伤后细胞死亡机制的动物模型的有用辅助手段。在此，我们描述一种新的体外模型，其中通过打孔装置在96孔微孔板的各个孔中产生28条平行切口来实现可重复的损伤水平。通过乳酸脱氢酶（LDH）测定或用于溴化乙锭同二聚体染色的定量荧光测定来测量细胞损失。神经胶质细胞培养显示细胞死亡局限于初始损伤部位，而神经元/神经胶质细胞培养显示细胞损失随时间有显著扩散。我们使用该模型来研究pH值和N-甲基-D-天冬氨酸（NMDA）受体在创伤后延迟损伤中的作用。用二氮嗪（MK-801）阻断NMDA受体或用针对NMDAR1的反义寡脱氧核苷酸处理具有神经保护作用。在酸性条件下观察到细胞死亡减少，而细胞外pH值升高与MK-801敏感的细胞损失增加有关。我们模型的优点包括：可重复诱导创伤；快速测量细胞损伤；以及使用96孔微孔板，这减少了时间和成本。该模型似乎非常适合研究创伤后神经元损伤的特定机制以及筛选潜在的神经保护剂。
