BrainPhys® increases neurofilament levels in CNS cultures, and facilitates investigation of axonal damage after a mechanical stretch-injury in vitro.
BrainPhys® increases neurofilament levels in CNS cultures, and facilitates investigation of axonal damage after a mechanical stretch-injury in vitro.
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DOI：
10.1016/j.expneurol.2017.11.013
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发表时间：
2018-03
影响因子：
5.3
通讯作者：
Kochanek PM
中科院分区：
文献类型：
--
作者：
Jackson TC;Kotermanski SE;Jackson EK;Kochanek PM
Neurobasal®/B27是用于体外研究原代神经元的金标准培养基。最近开发了一种替代培养基（BrainPhys®/SM 1），其相对于Neurobasal®或DMEM稳健地增强了神经元活性。据我们所知，尚未在神经元损伤的情况下探索BrainPhys®。在这里，我们表征了BrainPhys®在体外机械拉伸损伤模型中的效用。将原代大鼠皮质神经元维持在经典的Neurobasal®中，或依次维持在Neurocult®中，然后维持在BrainPhys®中（下文简称为“BrainPhys®维持的神经元”）。在体外第10天（DIV 10）比较了轴突标志物和参与神经传递的蛋白质的水平。与Neurobasal®中的神经元相比，BrainPhys®维持的神经元具有更高水平的GluN 2B、GluR 1、神经丝轻链/重链（NF-L & NF-H）和蛋白磷酸酶2A（PP 2A）。对BrainPhys®的机械拉伸损伤（50 ms/54%双轴拉伸）维持神经元适度地（尽管显著地）增加24小时乳酸脱氢酶（LDH）水平，但相对于未损伤的对照或给予较温和的38%拉伸损伤的神经元，损伤后轴突NF-L水平显著降低。此外，两次54%牵张损伤（串联）加剧了24小时LDH释放，增加了α-血影蛋白分解产物（SBDP），并降低了Tau水平。此外，与Neurobasal®中的神经元相比，BrainPhys®维持的培养物在单次54%拉伸损伤后24小时具有减少的细胞损伤标志物。最后，我们测试了慢病毒介导的促死亡蛋白RBM 5过表达加剧原代CNS培养物中神经元和/或轴突损伤的假设。在单次54%牵张损伤后，RBM 5过表达相对于空载体对照增加了24小时LDH释放和SBDP水平，但不影响NF-L水平或Tau。BrainPhys®是一种很有前途的新试剂，有助于研究体外轴突和/或神经元损伤中涉及的分子靶标。
Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury. Primary rat cortical neurons were maintained in classic Neurobasal®, or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as “BrainPhys® maintained neurons”). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B, GluR1, Neurofilament light/heavy chain (NF-L & NF-H), and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore, two 54% stretch-injuries (in tandem) exacerbated 24h LDH release, increased α-spectrin breakdown products (SBDPs), and decreased Tau levels. Also, BrainPhys® maintained cultures had decreased markers of cell damage 24h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally, we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24h LDH release, and SBDP levels, after a single 54% stretch-injury but did not affect NF-L levels or Tau. BrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro.
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