Continuous monitoring of caspase-3 activation induced by propofol in developing mouse brain
Continuous monitoring of caspase-3 activation induced by propofol in developing mouse brain
复制标题
DOI：
10.1016/j.ijdevneu.2016.04.007
复制
发表时间：
2016-06-01
影响因子：
1.8
通讯作者：
Iijima, Takehiko
中科院分区：
文献类型：
--
作者：
Konno, Ayumi;Nishimura, Akiko;Iijima, Takehiko
麻醉剂对发育中的大脑的神经毒性引起了麻醉科医生的注意。多项研究表明，大脑发育过程中接受麻醉会增强细胞凋亡。虽然细胞凋亡是神经网络成熟和脑功能整合之前发生的生理发育步骤，但病理损伤也涉及细胞凋亡。先前的研究表明，长时间接触麻醉剂会导致细胞凋亡。细胞凋亡级联到底何时在大脑中开始仍不确定。如果在麻醉早期就开始，即使是短期麻醉也会损害大脑。因此，应持续监测细胞凋亡，以阐明麻醉何时触发细胞凋亡级联反应。在这里，我们描述了使用体内模型检测 caspase-3 激活的连续监测系统的开发。使用出生后 0-4 天的杂合基因型 SCAT3 转基因小鼠 (n = 20) 的脑切片来监测 caspase-3 裂解。 SCAT3 是 ECFP 和 Venus 的融合蛋白，通过 caspase-3 可裂解肽 DEVD 连接。将海马 CM 部分的标本安装在共焦激光显微镜上，并连续灌注人工脑脊液、异丙酚（2,6-二异丙基苯酚，1 μM 或 10 μM）和二甲亚砜。每小时获取一次图像，持续五个小时。使用显示每个比率的像素数的直方图对 ECFP/金星比率图像进行像素分析。在 ECFP/Venus 比率的直方图中，比率 > 1 的区域表示来自 caspase-3 激活的 CA1 神经元的像素数。我们观察到直方图随着时间的推移向右移动，表明 caspase-3 激活。在异丙酚1μM组和10μM组中，这种右移在5小时时发生了显着变化，并且与对照组有明显不同。因此，实时荧光能量转移（FRET）成像能够识别新生儿脑切片中丙泊酚引发的细胞凋亡的发生。该模型可能是监测发育中大脑细胞凋亡的有用工具。 (C) 2016 ISDN。由爱思唯尔有限公司出版。保留所有权利。
The neurotoxicity of anesthetics on the developing brain has drawn the attention of anesthesiologists. Several studies have shown that apoptosis is enhanced by exposure to anesthesia during brain development. Although apoptosis is a physiological developmental step occurring before the maturation of neural networks and the integration of brain function, pathological damage also involves apoptosis. Previous studies have shown that prolonged exposure to anesthetics causes apoptosis. Exactly when the apoptotic cascade starts in the brain remains uncertain. If it starts during the early stage of anesthesia, even short-term anesthesia could harm the brain. Therefore, apoptogenesis should be continuously monitored to elucidate when the apoptotic cascade is triggered by anesthesia. Here, we describe the development of a continuous monitoring system to detect caspase-3 activation using an in vivo model. Brain slices from postnatal days 0-4 SCAT3 transgenic mice with a heterozygous genotype (n =20) were used for the monitoring of caspase-3 cleavage. SCAT3 is a fusion protein of ECFP and Venus connected by a caspase-3 cleavable peptide, DEVD. A specimen from the hippocampal CM sector was mounted on a confocal laser microscope and was continuously superfused with artificial cerebrospinal fluid, propofol (2,6-diisopropylphenol, 1 mu M or 10 mu M), and dimethyl sulfoxide. Images were obtained every hour for five hours. A pixel analysis of the ECFP/Venus ratio images was performed using a histogram showing the number of pixels with each ratio. In the histogram of the ECFP/Venus ratio, an area with a ratio > 1 indicated the number of pixels from caspase-3-activated CA1 neurons. We observed a shift in the histogram toward the right over time, indicating caspase-3 activation. This right-ward shift dramatically changed at five hours in the propofol 1 mu M and 10 mu M groups and was obviously different from that in the control group. Thus, real-time fluorescence energy transfer (FRET) imaging was capable of identifying the onset of apoptosis triggered by propofol in neonatal brain slices. This model may be a useful tool for monitoring apoptogenesis in the developing brain. (C) 2016 ISDN. Published by Elsevier Ltd. All rights reserved.