Traumatic brain injury: early mechanosensitive events in astrocytes

描述（由申请人提供）：创伤性脑损伤（TBI）是由外部机械力（如爆炸或碰撞）引起的脑损伤。我们目前对创伤性脑损伤的理解主要来自体内研究，这些研究显示创伤性脑损伤后取样的细胞有可测量的生物学效应。在损伤刺激过程中，脑细胞的主要机械应力以及它们如何被转导成涉及TBI的细胞和分子事件，目前所知甚少。本研究旨在研究哪些刺激特性对细胞损伤最为关键，以及机械刺激如何与细胞次生过程耦合。我们将在一个由快速压力伺服驱动的微流体室中使用组织培养的成年星形胶质细胞来产生随时间变化的流体剪切，可以模拟爆炸产生的剪切应力。我们将使用遗传编码的荧光应力传感器直接测量特定细胞骨架蛋白中的应力。这项研究有两个具体目的。目的1确定导致长期改变的刺激物的物理性质。通过使用不同振幅、上升时间和频率的冲击波测量特定细胞骨架蛋白的应力时间过程，我们将确定导致不可逆变形的参数。这种不可逆性将通过研究解剖结构和应力分布的变化来确定。目的2研究细胞在机械损伤期间和之后的反应，以确定机械刺激如何改变细胞的稳态。为此，我们将测量细胞内Ca2+和细胞体积的时间依赖性变化。这些特性将与细胞应力同时测量，以确定因果关系。我们将使用特异性抑制剂GsMTx4进一步测试机械敏感通道通过Ca2+的作用，并探讨GsMTx4是否可以用作预防治疗。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is brain damage resulting from an external mechanical force, such as blast or crashes. Our current understanding of TBI is derived mainly from in vivo studies that show measurable biological effects on cells sampled after TBI. Little is known about the primary mechanical stresses in brain cells during damaging stimuli, and how they are transduced into cellular and molecular events involved in TBI. This proposal aims to examine what stimulus properties are most critical to cell injury and how the mechanical stimulus is coupled to secondary cellular processes. We will use tissue cultured adult astrocytes in a microfluidic chamber driven by a fast pressure servo to generate time dependent fluid shear that can be made to emulate shear stress from a blast. We will directly measure the stresses in specific cytoskeletal proteins using genetically encoded fluorescent stress sensors. The research has two specific aims. Aim 1 identifies physical properties of the stimulus that lead to long term alterations. By measuring the time course of stresses in specific cytoskeletal proteins using shockwaves of various amplitude, rise time, and frequency, we will determine the parameters that lead to irreversible deformation. This irreversibility will be determined by investigating changes in anatomy and stress distribution. Aim 2 investigates the cells' response during and immediately after mechanical insult to determine how mechanical stimulus alters cell homeostasis. For this we will measure the time dependent changes of intracellular Ca2+ and cell volume. These properties will be measured simultaneously with cellular stress to determine causality. We will further test the role of mechanosensitive channels for passing Ca2+ using specific inhibitor, GsMTx4, and explore whether pre-administration of GsMTx4 might be used as a preventive therapy.