Isolation of cerebrospinal fluid from rodent embryos for use with dissected cerebral cortical explants.
Isolation of cerebrospinal fluid from rodent embryos for use with dissected cerebral cortical explants.
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DOI：
10.3791/50333
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发表时间：
2013-03
期刊：
影响因子：
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通讯作者：
Mauro W. Zappaterra;A. LaMantia;C. Walsh;Maria K. Lehtinen
中科院分区：
文献类型：
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作者：
Mauro W. Zappaterra;A. LaMantia;C. Walsh;Maria K. Lehtinen
CSF是一种复杂的液体，在整个发育过程中和成年期具有动态变化的蛋白质组。在胚胎发育过程中，新生CSF在前神经管闭合时与羊水分化。随后几天，随着脑室和脉络丛的神经上皮祖细胞产生CSF，CSF体积增加。胚胎CSF接触发育中的脑和脊髓的神经干细胞的顶室表面。CSF为发育中的大脑的扩张提供关键的流体压力，并以时间特异性的方式将重要的生长促进因子分配给神经祖细胞。为了研究CSF的功能，分离不受血液或发育中的端脑组织污染的胚胎CSF纯样品非常重要。在这里，我们描述了一种技术，以分离相对纯净的心室胚胎CSF样品，可用于广泛的实验测定，包括质谱，蛋白电泳，细胞和原代外植体培养。我们演示了如何在多孔聚碳酸酯膜上解剖和培养皮质外植体，以便在培养基或CSF体积减少的情况下生长发育中的皮质组织。通过这种方法，可以使用来自不同年龄或条件的CSF进行实验，以研究CSF蛋白质组对靶细胞的生物活性。
The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.