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首页> 外文会议>Conference on Multiphoton Microscopy in the Biomedical Sciences IV; 20040125-20040127; San Jose,CA; US >Multiphoton Tissue FRET Demonstrates in vivo BAD/Bcl-xL Heterodimerization in Injured Axons Following Traumatic Brain Injury in Rats
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Multiphoton Tissue FRET Demonstrates in vivo BAD/Bcl-xL Heterodimerization in Injured Axons Following Traumatic Brain Injury in Rats

机译：多光子组织FRET证实大鼠脑外伤后轴突体内BAD / Bcl-xL异二聚化。

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摘要

Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. A devastating element of TBI is the diffuse and widespread injury of axons in a process known as traumatic axonal injury (TAI). TAI is a difficult entity to study as gross protein or molecular analyses have been largely precluded due to the requisite for specifically localizing protein changes to axons rather than the supporting glia or neuronal soma. As such, much of the mechanistic insight into TAI pathogenesis thusfar has come through histochemical and immunohistochemical analyses. In order to address the next generation of questions in TAI pathogenesis it has become critical to develop methodologies that allow for direct examination of protein-protein interactions in relation to sites of TAI. In the current communication, we report on a modified method of multiphoton Fluorescent Resonance Energy Transfer (FRET) microscopy that allows for the direct assessment of protein-protein interactions in aldehyde fixed tissue sections through the use of a conventional dual-label immunofluorescent approach. In the utilization of this technique, we explored whether the bcl-2-related proteins BAD and Bcl-xL heterodimerize in a pro-apoptotic fashion within traumatically injured axons following TBI. Adult SD rats were subjected to impact acceleration TBI and euthanized at multiple time points. Vibratome sections derived from adult SD rats that had previously undergone impact acceleration TBI were processed for immunohistochemical double labeling with anti-BAD 1° antibody/Alexa 488 2°antibody, followed by anti-Bcl-xL 1°antibody/Alexa 555 2°antibody. Images were processed for spectral bleedthrough, and efficiency/distance calculations were performed. BAD/Bcl-xL heterodimerization was examined in relation to cytochrome c release and caspase-3 activation, by employing a third immunofluorescent label visualized with an Alexa 647 dye. Multiphoton FRET microscopy was carried out upon 40 micron thick sections. Further, to determine if FRET analysis could be performed in thick tissue specimens, 100 and 200 micron thick sections were examined as well. At 6h postinjury, swollen axons in medial lemniscus demonstrated a mean energy transfer efficiency greater than 20% indicating formation of Bad-Bcl-xL complexes. Thick tissue specimens up to 200 microns thick likewise demonstrated FRET efficiencies greater than 20%. Specimens positively labeled for either cytochrome c or caspase-3 demonstrated FRET efficiencies greater than 10%. The current investigation demonstrates FRET microscopy can be employed to assess protein-protein interactions in aldehyde-fixed tissue sections through multi-label immunofluorescent methodologies. It is believed that the current approach will have widespread applicability as examination of protein-protein interactions within in vivo systems may now be assessed on the cellular and subcellular level within aldehyde fixed tissue sections.
机译：在西方世界，创伤性脑损伤（TBI）仍然是45岁以下人群最常见的死亡原因。 TBI的毁灭性因素是轴突的弥漫性和广泛性损伤，此过程称为创伤性轴突损伤（TAI）。由于需要将蛋白质变化专门定位在轴突而不是支持神经胶质细胞或神经体上，因此TAI很难研究，因为总蛋白或分子分析已被大大地排除在外。因此，迄今为止，对TAI发病机理的许多机理研究都是通过组织化学和免疫组织化学分析得出的。为了解决TAI发病机理中的下一代问题，开发允许直接检查与TAI位点有关的蛋白质-蛋白质相互作用的方法已变得至关重要。在当前的通讯中，我们报告了一种改进的多光子荧光共振能量转移（FRET）显微镜方法，该方法允许通过使用常规的双标记免疫荧光方法直接评估醛固定组织切片中的蛋白质-蛋白质相互作用。在这项技术的利用中，我们探讨了TBI后受创伤的轴突内bcl-2相关蛋白BAD和Bcl-xL是否以促凋亡的方式异源二聚体。成年SD大鼠经过冲击加速TBI并在多个时间点安乐死。用抗BAD 1°抗体/ Alexa 488 2°抗体，然后是抗Bcl-xL 1°抗体/ Alexa 555 2°抗体对源自成年SD大鼠的先前曾经历过冲击加速TBI的玻璃体切片进行免疫组织化学双标记处理。 。处理图像以进行光谱渗漏，并进行效率/距离计算。 BAD / Bcl-xL异二聚化与细胞色素c释放和caspase-3激活有关，通过使用用Alexa 647染料可视化的第三个免疫荧光标记进行了检查。在40微米厚的切片上进行了多光子FRET显微镜检查。此外，为了确定是否可以在厚组织样本中进行FRET分析，还检查了100和200微米厚的切片。受伤后6小时，内侧红膜中轴突肿胀表明平均能量转移效率大于20％，表明形成了Bad-Bcl-xL复合物。同样厚至200微米的厚组织样本也显示出FRET效率大于20％。阳性标记的细胞色素c或caspase-3的标本显示FRET效率大于10％。目前的研究表明，FRET显微镜可用于通过多标记免疫荧光方法评估醛固定组织切片中的蛋白质-蛋白质相互作用。据信，当前的方法将具有广泛的适用性，因为现在可以在醛固定组织切片内的细胞和亚细胞水平上评估体内系统内的蛋白质-蛋白质相互作用。

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